Modulators of retinoid-related orphan receptor gamma

ABSTRACT

Methods for modulating (inhibiting or stimulating) retinoid-related orphan receptor γ (RORγ) activity. This modulation has numerous effects, including inhibition of TH-17 cell function and/or TH-17 cell activity, and inhibition of re-stimulation of TH-17 cells, which are beneficial to treatment of inflammation and autoimmune disorders. Stimulation of RORγ results in stimulation of TH-17 cell function and/or activity which is beneficial for immune-enhancing compositions (e.g., vaccines).

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/592,692, which is a continuation of U.S. application Ser. No.13/766,076, filed Feb. 13, 2013 (now U.S. Pat. No. 9,657,053), which isa continuation of U.S. application Ser. No. 13/114,616, filed May 24,2011 (now U.S. Pat. No. 8,389,739), which is a continuation of U.S.application Ser. No. 11/867,637, filed Oct. 4, 2007, which claimspriority under 35 U.S.C. § 119(e) to U.S. Provisional Application No.60/849,903, filed Oct. 5, 2006. The contents of these applications areincorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Numbers 1R43 AI060447-01, 1 R03 NS 050879-01, 1 R43 DK071461-01, 1 R43MH075461-01, 1 R43 NS 059219-01, 1 R43 CA099875-01A1, and 1 R43AR055427-01 from the National Institutes of Health. The government hascertain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledORPH-001C4_SequenceListing.txt, created May 9, 2018, which is 12.5 kbbytes in size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods for modulating (inhibiting orstimulating) retinoid-related orphan receptor γ (RORγ) activity. Thismodulation has numerous effects, including inhibition of T_(H)-17 cellfunction and/or T_(H)-17 cell activity, and inhibition of re-stimulationof T_(H)-17 cells, which are beneficial to treatment of inflammation andautoimmune disorders. In contrast, stimulation of RORγ results instimulation of T_(H)-17 cell function and/or activity which isbeneficial for immune-enhancing compositions (e.g., vaccines). Morespecifically, the present invention relates to methods for inhibitingdifferentiation of T cells into T_(H)-17 cells, or inhibiting theactivity of T_(H)-17 cells and other T cells that express IL-17, bycontacting a population of T cells that may include T_(H)-17 cells, withan antagonist of RORγ.

BACKGROUND OF THE INVENTION

Many forms of serious human disease result from an autoimmune attack onthe body. Many forms of autoimmune disease, such as rheumatoidarthritis, multiple sclerosis, psoriasis, and Crohn's disease, areparticularly difficult to treat. An activated CD4⁺ T cell that releasesinterleukin-17 (IL-17), a powerful pro-inflammatory cytokine (Aggarwal,Ghilardi et al. 2003), and is referred to as a T_(H)-17 cell (Bettelli,Carrier et al. 2006; Mangan, Harrington et al. 2006; Veldhoen, Hockinget al. 2006), may be a significant pathogenic factor in autoimmunedisease, based on observations that patients have high levels of IL-17expression in target tissues. For example, the level of IL-17 mRNA wasfound to be elevated in brain autopsy tissue from patients with MScompared with controls, and the number of IL-17 positive mononuclearcells was also increased in the cerebrospinal fluid of affected patients(Matusevicius, Kivisakk et al. 1999; Lock, Hermans et al. 2002;Vaknin-Dembinsky, Balashov et al. 2006). Rheumatoid arthritis (RA) alsoappears to involve a T cell-mediated autoimmune reaction. The level ofIL-17 mRNA in synovial fluid of rheumatoid arthritis patients ispredictive of disease progression (Kirkham, Lassere et al. 2006).Furthermore, T_(H)-17 cells have been isolated from intestinal biopsiesof patients with Crohn's Disease (Annunziato, Cosmi et al. 2007), andtheir frequencies are elevated in comparison to T cells isolated fromnormal intestine or peripheral blood. In disease tissue of psoriasispatients, levels of IL-22, another proinflammatory cytokine produced byT_(H)-17 cells, are elevated (Zheng, Danilenko et al. 2007).

T_(H)-17 cells, also referred to as IL-17⁺CD4⁺ T or T_(IL-17) cells,have only been recently characterized and are distinct from the T_(H)1and T_(H)2 lineages of CD4⁺ effector T cells. The relationship ofT_(H)-17 cells to the T_(H)1 and T_(H)2 lineages is diagrammed inFIG. 1. T_(H)1 and T_(H)2-derived cytokines (such as IFN-γ and IL-4)block T_(H)-17 formation (FIG. 1) and targeted deletion of sometranscription factors considered to be central for maintenance of theT_(H)1 and T_(H)2 phenotype have no major effect on T_(H)-17differentiation (Harrington, Hatton et al. 2005; Park, Li et al. 2005).IL-23 expression in mouse is required for induction of T cell-derivedIL-17, and in culture IL-23 seems to act as a survival factor forpre-existing T_(H)-17 cells but does not stimulate T_(H)-17 formationfrom naïve CD4⁺ T cells. Instead, the combination of TGFβ and IL-6 isrequired to stimulate the formation of T_(H)-17 cells from naive mouseCD4⁺ T cells, and there is genetic evidence in mouse for the involvementof IL-6 and TGFβ in T_(H)-17 formation in vivo (Bettelli, Carrier et al.2006; Mangan, Harrington et al. 2006; Veldhoen, Hocking et al. 2006).The cytokine requirements for T_(H)-17 diferentiation in human aresomewhat different that in mouse but the T_(H)-17 phenotype is similar,including expression of IL-17, IL-22, and RORγ (Annunziato, Cosmi et al.2007; Kebir, Kreymborg et al. 2007).

The older view that autoreactive CD4⁺ T_(H)1 cells are a prime cause forthe development or progression of MS and other forms of autoimmunedisease has been challenged (Steinman 2007) by recent results fromrodent models showing that mice with a targeted deletion in the p35subunit of IL-12, which is required for the formation of T_(H)1 cells,are still highly susceptible to the induction of experimental autoimmuneencephalomyelitis (EAE) after immunization with myelin antigens (Becher,Durell et al. 2002; Gran, Zhang et al. 2002). In contrast, the knockoutof either p19, the unique subunit of the cytokine IL-23, or p40, acommon subunit of the two cytokines IL-12 and IL-23, create mice thatare resistant to the induction of EAE (Cua, Sherlock et al. 2003;Langrish, Chen et al. 2005).

In addition to EAE, development of disease in a mouse model ofrheumatoid arthritis, collagen-induced arthritis (Courtenay, Dallman etal. 1980), is also dependent on IL-23 function and is correlated with anincreased level of T_(H)-17 cell activity, such as release of IL-17 inaffected tissues (Murphy, Langrish et al. 2003; Sato, Suematsu et al.2006). Inhibition of IL-17 may be a viable alternative to inhibition ofTNFα in treatment of RA (Lubberts, Koenders et al. 2005). Further, IL-23expression is required for induction of IBD in mouse, and T_(H)-17 cellsappear to be an important downstream mediator of IL-23 effect in thismodel (Yen, Cheung et al. 2006). Disease progression in EAE models ispartially suppressed in IL-17^(−/−) mice or after treatment withanti-IL-17 antibodies (Iwakura and Ishigame 2006). IL-17^(−/−) mice alsoshow reduced incidence and severity in a collagen-induced arthritis(CIA) model for rheumatoid arthritis and IL-17 antibodies have beenshown to attenuate development of intestinal inflammation in rodentmodels of IBD (Nakae, Nambu et al. 2003; Yen, Cheung et al. 2006).Further, purified autoreactive T_(H)-17 cells strongly induceencephalomyelitis when transferred to naive mice, leading to moredramatic disease manifestation compared to disease induction by T_(H)1CD4⁺ T cells (Langrish, Chen et al. 2005; Komiyama, Nakae et al. 2006).The IL-27 receptor is prominently expressed in T_(H)-17 cells and theaction of IL-27 as an inhibitor of murine EAE is correlated to reducedT_(H)-17 cell number in the animal (Batten, Li et al. 2006; Stumhofer,Laurence et al. 2006). Therefore, the activity of murine T_(H)-17 cellsis well correlated to disease.

Inhibition of the human equivalent of the mouse T_(H)-17 cell is ahighly desirable target for new therapeutic agents to treat autoimmunedisease. Recent findings implicate T_(H)-17 cells in the pathogenesis ofCrohn's disease and more generally inflammatory bowel disease (IBD).Antibody to the common subunit of IL-23 and IL-12, p40, is safe and maybe clinically effective in treatment of Crohn's disease (Mannon, Fuss etal. 2004). A recent Phase 2 clinical trial also shows that anti-p40 ishighly effective in the treatment of psoriasis (Krueger, Langley et al.2007). The identification of T_(H)-17 cells has not only providedinsight into autoimmune pathogenesis, it has also revealed a majorpathway of adaptive immunity for extracellular microbes (Mangan,Harrington et al. 2006; Annunziato, Cosmi et al. 2007). Stimulation ofT_(H)-17 differentiation, function and cytokine release therefore hasthe potential to enhance protective immunity by increasing T cellreactivity to pathogenic organisms and other targets, such ascancer-associated antigens.

Structure and Function of RORγ

RORγ (NR1F3), a ligand-regulated nuclear transcription factor from thesteroid/retinoid/thyroid family (Jetten, Kurebayashi et al. 2001), hasbeen shown to be essential for CD4⁺ T_(H)-17 development and/orfunction. RORγ participates in and is required for the development ofT_(H)-17 cells. T_(H)-17 cells are absent from genetically-engineeredmice that fail to express a specific splicing isoform of RORγ, RORγt(Ivanov, McKenzie et al. 2006; Littman and Eberl 2006). Furthermore, anRORγt-GFP transgene that expresses GFP from the RORγt promoter isexpressed in CD4⁺IL-17⁺ T cells from the lamina propria of the gut andother tissues. In cell culture, transfection of RORγt into naïve murineCD4⁺ T cells induces differentiation of these cells into IL-17expressing T cells even in the absence of the inducing cytokines IL-6and TGFβ. The data suggest that the transcriptional activity of RORγt isof major importance to T_(H)-17 cell differentiation and function(Ivanov, McKenzie et al. 2006). RORγt expression is induced in thepresence of TGFβ and IL-6 or by TGFβ and IL-21, an autocrine cytokinereleased from developing T_(H)-17 cells in response to IL-6 (Ivanov,McKenzie et al. 2006; Korn, Bettelli et al. 2007; Nurieva, Yang et al.2007; Zhou, Ivanov et al. 2007).

The expression of RORγ follows a similar pattern in human as in mouse Tcells: RORγ is more highly expressed in IL-17 or IL-17/IFNγ expressingCD4⁺ T cells (Th-17) than in CD4⁺ T cells that express IFNγ alone (Th-1)(Acosta-Rodriguez, Napolitani et al. 2007; Acosta-Rodriguez, Rivino etal. 2007; Annunziato, Cosmi et al. 2007; Wilson, Boniface et al. 2007).Finally, it has been reported that other murine T cell types expressRORγt, including γδTCR⁺ cells, CD8⁺ T cells, and iNKT cells (Ivanov,McKenzie et al. 2006; Ivanov and Littman 2007). Since these cells arealso express IL-17, it is possible that RORγt is required for thedifferentiation of the IL-17⁺ subpopulations of several other types of Tcells.

Finally, in the absence of RORγt, mice are much less susceptible to theinduction of EAE (Ivanov, McKenzie et al. 2006). These studies werecarried out in immunodeficient mice. RORγt^(−/−) mice lack lymph notes,and although they are resistant to the development of EAE, a furtherstudy was carried out by adoptive transfer of RORγt wild type orRORγt^(−/−) bone marrow to immunodeficient mice. While transfer ofnormal bone marrow rendered the mice sensitive to the induction of EAEby peptide immunization, the RORγt^(−/−) transfectants weresubstantially resistant (Ivanov, McKenzie et al. 2006). These datasuggest that RORγt mediated regulation of T cell differentiation isinvolved in the development of autoimmune disease.

RORγ Structure.

RORγ, like other members of the nuclear receptor family, has a bipartitestructure with two major functional domains, a DNA binding domain (DBD)and a ligand binding domain (LBD, see FIG. 2) (Medvedev, Yan et al.1996). Ligand-regulated transcription of the nuclear receptors ismediated through the LBD, which has been crystallized for many receptors(Li, Lambert et al. 2003), including RORα and RORβ, the receptors mostclosely related to RORγ. The RORγ LBD is predicted to have a bindingpocket similar to RORα and RORβ (Stehlin, Wurtz et al. 2001; Kallen,Schlaeppi et al. 2004). The retinoic acid receptors (RARα, RARβ, andRARγ) are more distantly related and appear not to have a functionaloverlap with the RORs (Jetten, Kurebayashi et al. 2001).

The nuclear receptor LBD recruits transcriptional coregulators (FIG. 2)in response to small molecule compounds (Li, Lambert et al. 2003; Savkurand Burris 2004). These coregulatory proteins act as sensors for theconformational state of the ligand-bound complex and in turn regulatethe recruitment of transcriptional factors to chromatin adjacent to thereceptor. Short peptide domains of the coregulatory proteins requiredfor interaction with the nuclear receptor contain the conserved sequenceLXXLL (SEQ ID NO: 1), and synthetic peptide recruitment assays based onthis motif are widely used to monitor the binding of agonists andantagonists to the nuclear receptor LBD (Lee, Elwood et al. 2002; Savkurand Burris 2004), including for an assay described herein for RORγ.

Functional Studies of RORγ.

Recognition of specific DNA motifs by the nuclear receptor DBDdetermines specificity for gene transcription; however, little is knownabout specific gene targets for RORγ, and the major findings on RORγfunction have been derived from studies of gene-targeted mice(Kurebayashi, Ueda et al. 2000; Sun, Unutmaz et al. 2000; Eberl andLittman 2004; Eberl, Marmon et al. 2004). RORγ has two splicingisoforms, RORγ and RORγt (He, Deftos et al. 1998). RORγt differs fromRORγ by a truncation of 21 amino acids at the N-terminal and is theisoform specifically expressed in thymus, lymph node precursors, andT_(H)-17 cells (Eberl and Littman 2004; Eberl, Marmon et al. 2004;Ivanov, McKenzie et al. 2006; Littman and Eberl 2006), the major tissuesaffected in knockout studies of RORγ. The significance of the N-terminaldeletion of RORγ is not known, but it is unlikely to affect ligandspecificity or LBD function, which is encoded at the receptor'sC-terminal and is identical in the two splicing isoforms.

In addition to its requirement for T_(H)-17 differentiation, RORγ hasother discrete functions in the immune system. In its absence, thesurvival of the major subtype of developing T lymphocytes, the CD4⁺CD8⁺double positive (DP) thymocytes, is reduced (Kurebayashi, Ueda et al.2000; Sun, Unutmaz et al. 2000), and the embryonic formation of lymphnodes and Peyer's patches is blocked (Eberl, Marmon et al. 2004). RORγtis an early marker for the embryonic formation of lymphoid tissueinducer or LTi cells. LTi cells are involved in lymph node and Peyer'spatch formation during embryogenesis (Eberl, Marmon et al. 2004). Afterbirth, an LTi-like cell participates in formation of intermediatelymphoid follicles (ILFs) of the gut. These lymphoid structures appearto participate in the gut immune response (Eberl and Littman 2004). RORγis also expressed in liver, muscle, and fat (Jetten, Kurebayashi et al.2001; Fu, Sun et al. 2005). A recent study of RORγ^(−/−) mice alsosuggests that the receptor has some regulatory effects on Phase 1 andPhase 2 detoxification enzymes (Kang, Angers et al. 2007).

Clinical Significance.

Three major points of action of RORγ in the immune system have beenidentified in gene knockout studies; T cells, including the T_(H)-17cell, lymph node formation, and survival of DP thymocytes. Of these,regulation of IL-17 T cell function, including T_(H)-17 differentiation,appears to be most relevant to human therapeutics. Not only is thereceptor absolutely required for T_(H)-17 differentiation, but thesupply of pathogenic T_(H)-17 cells must be constantly replenished sincethey are destroyed in target tissue (Gold, Linington et al. 2006).Inhibition of new T_(H)-17 formation will therefore have importantbenefits. RORγ is expressed in human memory T_(H)-17 cells(Acosta-Rodriguez, Rivino et al. 2007; Annunziato, Cosmi et al. 2007),and data presented herein shows that RORγ antagonists block IL-17expression in human peripheral blood mononuclear cells (PBMCs). Theprimary source of IL-17 in PBMCs has been reported to be memory T cells(Shin, Benbernou et al. 1999). Finally, RORγ enhances, but is notabsolutely required for, the survival of the major developing T celltype of the thymus, the DP thymocyte. In the knockout animal, there isno evidence of immunodeficiency although splenic and peripheralfrequencies of some immune cell types are changed. However, there is amuch higher rate of apoptosis among thymocytes and thymocyte number isreduced (Kurebayashi, Ueda et al. 2000; Zhang, Guo et al. 2003). Thethymus of an adult has already atrophied to a considerable degree andcan be reversibly suppressed by many forms of pharmacological treatment,including exposure to steroids (Haynes, Markert et al. 2000). These datasuggest that secondary effects of an RORγ antagonist on thymic functionin human may not be clinically significant.

Prior to 1990, no drugs to treat MS were available. In the last 15years, several treatment options have emerged, primarily various formsof INFβ (Avonex, Rebif, Betaseron), Glatiramer acetate (Copaxone) andthe chemotherapeutic drug mitoxantrone (Novantrone) (Rolak 2003). INFβand Glatiramer acetate both appear to inhibit T cell activation and bothdrugs reduce the number of attacks in relapsing-remitting MS but havelittle effect in the progressive phase of the disease (Dhib-Jalbut 2002;Rolak 2003). The long-term benefits of these drugs are unclear.Mitroxantrone appears to retard progression and delay disability insecondary progressive MS. However, toxicity of this drug is verylimiting and Mitroxantrone is considered a short-term treatment option.More recently, a humanized antibody to α4β1 integrin (NATALIZUMAB,Tysabri) has been approved for the treatment of MS and has been shown toslow disease progression and reduce relapse rate in several clinicaltrials using different outcome measures (Steinman 2005). Compared toexisting treatments, the efficacy of Tysabri is quite dramatic. However,several cases of progressive multifocal leukoencephalopathy (PML), alethal resurgence of a latent viral infection linked to theimmunosuppressive action of the drug, led to withdrawal (Rudick, Stuartet al.). Tysabri has now been reintroduced into the market with muchstricter patent monitoring. Tysabri likely blocks both T_(H)-17 andT_(H)1 cells. A novel small molecule drug that is specific for T_(H)-17cells and does not compromise the antiviral activity of T_(H)1 cellscould be safer and more effective for several reasons: (1) a smallmolecule drug, administered daily, can be rapidly withdrawn ifsignificant side effects occur; (2) a small molecule drug is morereadily manufactured and more easily administered than a biologic, suchas Tysabri; and (3) T_(H)1 cells suppress T_(H)-17 differentiation andthus specific inhibition of T_(H)-17 cells may be more effective.

A number of other small molecule drugs are marketed for autoimmunediseases such as rheumatoid arthritis, IBD, and psoriasis. Many ofthese, such as methotrexate or azathioprine, carry significant toxicitybecause of anti-metabolite or anti-mitotic effects. Dosing is usuallylimited. A small molecule drug that has a more specific mechanism ofaction, such as inhibition of T_(H)-17 cells or other IL-17 expressing Tcells, are likely to be safer and, hence, more efficacious as dosagesmay be elevated to have a substantial inhibitory effect on target cells.

Pharmacologically useful ligands to members of the ROR family of orphannuclear receptors have not been identified in the published literature.Cholesterol and cholesterol sulfate occupy the ligand binding pocketwithin the receptor LBD of RORα as determined by x-ray crystallography(Kallen, Schlaeppi et al. 2002; Kallen, Schlaeppi et al. 2004), but, dueto the fact that these molecules are plentiful in normal cells, it hasnot been possible to use these molecules in order to characterize RORαas a pharmacological target (Moraitis and Giguere 2003). A series ofRORα ligands was published in 1996 (Missbach, Jagher et al. 1996;Wiesenberg, Chiesi et al. 1998), but these findings have not beenindependently replicated or evaluated by functional criteria describedbelow. One of the proposed ligands for RORβ, melatonin, has beenchallenged in the literature (Becker-Andre, Wiesenberg et al. 1994;Greiner, Kirfel et al. 1996; Becker-Andre, Wiesenberg et al. 1997). Morerecently, it was proposed that all-trans retinoic acid and the syntheticretinoid ALRT 1550 are functional ligands for RORβ and that these twoligands also regulate RORγ in a similar manner, in both cases inhibitingthe transcriptional activity of the receptors. All-trans retinoic acidand ALRT 1550 were referred to as “functional” ligands because they werepresumed to both bind and regulate transcription through RORβ. Theligands are unlikely to be useful pharmacologically because they arepotent activators of the retinoid receptors, RARα, RARβ, RARγ (Thacher,Vasudevan et al. 2000). Therefore all of these ligands fail the test offunctional usefulness either on the criterion that their effects havenot been reproducible, or because they are ubiquitous, or because theylack specificity. This application describes assay for RORγ, and RORγligands that have pharmacologically useful potency (in the range of 50nM to 1 μM), that have good selectivity, as demonstrated by assays forother members of the nuclear receptor family. These ligands, bothagonists and antagonists, have drug-like properties as indicated byrational structure activity relationships among analogues as well asother drug-like properties such as bioavailability and activity incellular and animal models. Specific RORγ antagonists are predicted tobe highly useful in treatment of autoimmune disease by blocking T_(H)-17function. Agonists of RORγ are predicted to enhance immunity and to haveapplication in stimulation of vaccination and in adjuvant cancertherapy.

SUMMARY OF THE INVENTION

The present invention provides a method of inhibiting T_(H)-17 celldifferentiation from naïve T cells, or T_(H)-17 cell function/activity,comprising contacting a population of T cells that may include T_(H)-17cells with an effective amount of an antagonist of retinoic acid relatedorphan receptor γ (RORγ). In one embodiment, T_(H)-17 cellfunction/activity is release of a cytokine. The cytokine may beinterleukin-17 or interleukin-22. In one embodiment, the RORγ antagonistis at least 20-fold more potent as an RORγ antagonist than as an LXRagonist. In another embodiment, the T_(H)-17 cell differentiation orcytokine release is associated with an inflammatory or an autoimmunedisorder. In another embodiment, the inflammatory or autoimmune disorderis arthritis, diabetes, multiple sclerosis, uveitis, rheumatoidarthritis, reactive arthritis, sarcoidosis, psoriasis, psoriaticarthritis, asthma, bronchitis, allergic rhinitis, chronic obstructivepulmonary disease, atherosclerosis, H. pylori infections, ulcersresulting from H. pylori infections, inflammatory bowel disease, Crohn'sDisease ulcerative colitis or sprue. Preferably, the RORγ antagonist isa small molecule drug, and is preferably not a polynucleotide. In oneembodiment, the antagonist has the structure:

wherein R₁═H, C₁-C₆ alkyl, F, Cl, Br, I, NO2; R₂═C₁-C₄ alkyl; and X═OH,or a pharmaceutically acceptable salt, prodrug, derivative or metabolitethereof.

In one embodiment, the antagonist is OR-1050. In another embodiment, theantagonist has the structure:

wherein X═O or is absent (X═H,H), or a pharmaceutically acceptable salt,prodrug, derivative or metabolite thereof.

In other embodiments, the antagonist is OR-885, OR-345, OR-13571,OR-2161, OR-133171, or a pharmaceutically acceptable salt, prodrug,derivative or metabolite thereof.

The present invention also provides a method of inhibiting T_(H)-17 celldifferentiation from naïve T cells, or T_(H)-17 cell function/activity,in an individual in need thereof, comprising administering an effectiveamount of an RORγ antagonist to the individual. In one embodiment, theT_(H)-17 cell function/activity is release of a cytokine. In oneembodiment, the cytokine is interleukin-17 or interleukin-22. In anotherembodiment, the RORγ antagonist is at least 20-fold more potent as RORγantagonist than as LXR agonist. In one embodiment, the RORγ antagonistis orally administered. In another embodiment, the RORγ antagonist isadministered on a daily basis without resulting in weight loss orhypertriglyceridemia. In another embodiment, the T_(H)-17 celldifferentiation or cytokine release is associated with an inflammatoryor autoimmune disorder. In another embodiment, the inflammatory orautoimmune disorder is arthritis, diabetes, multiple sclerosis, uveitis,rheumatoid arthritis, reactive arthritis, sarcoidosis, psoriasis,psoriatic arthritis, asthma, bronchitis, allergic rhinitis, chronicobstructive pulmonary disease, atherosclerosis, H. pylori infections,ulcers resulting from H. pylori infections or inflammatory boweldisease, Crohn's Disease, ulcerative colitis, or sprue. In oneembodiment, the antagonist has the structure:

wherein R₁═H, C₁-C₆ alkyl, F, Cl, Br, I, NO2; R₂═C₁-C₄ alkyl; and X═OH,or a pharmaceutically acceptable salt, prodrug, derivative or metabolitethereof.

In one embodiment, the antagonist is OR-1050. In another embodiment, theantagonist has the structure:

wherein X═O or is absent (X═H,H), or a pharmaceutically acceptable salt,prodrug, derivative or metabolite thereof.

In another embodiment, the antagonist is OR-885, OR-345, OR-13571,OR-2161, OR-133171 or a pharmaceutically acceptable salt, prodrug,derivative or metabolite thereof.

The present invention also provides a method of treating an inflammatoryor autoimmune disease in an individual, comprising identifying anindividual in need of such treatment, and administering an effectiveamount of an RORγ antagonist the individual. In another embodiment, theRORγ antagonist is at least 20-fold more potent as RORγ antagonist thanas LXR agonist. In one embodiment, the RORγ antagonist is orallyadministered. In another embodiment, the RORγ antagonist is administeredon a daily basis without resulting in weight loss orhypertriglyceridemia. In one embodiment, the inflammatory or autoimmunedisorder is arthritis, diabetes, multiple sclerosis, uveitis, rheumatoidarthritis, reactive arthritis, sarcoidosis, psoriasis, psoriaticarthritis, asthma, bronchitis, allergic rhinitis, chronic obstructivepulmonary disease, atherosclerosis, H. pylori infections, ulcersresulting from H. pylori infections or inflammatory bowel disease. Inone embodiment, the inflammatory bowel disease is Crohn's disease,ulcerative colitis or sprue. Preferably, the RORγ antagonist is a smallmolecule drug. It is preferably not a polynucleotide, including DNA,antisense, siRNA, and the like. In one embodiment, the antagonist hasthe structure:

wherein R1=H, C1-C6 Alkyl, F, Cl, Br, I, NO2; R2=C1-C4 Alkyl; and X═OH,or a pharmaceutically acceptable salt, prodrug, derivative or metabolitethereof.

In one embodiment, the antagonist is OR-1050. In another embodiment, theantagonist has the structure

wherein X═O or is absent (X═H,H), or a pharmaceutically acceptable salt,prodrug, derivative or metabolite thereof. In other embodiments, theantagonist is OR-885, OR-345, OR-13571, OR-2161, OR-133171 or apharmaceutically acceptable salt, prodrug, derivative or metabolitethereof.

The present invention also provides a method of screening for agonistsor antagonists to RORα, RORγ or RORβ, comprising contacting a compoundwith a labeled, expressed ROR LBD and a labeled peptide that includesresidues 710-720 (RTVLQLLLGNP; SEQ ID NO: 2) of human RIP140; andmeasuring the proximity of the two labels, wherein binding of labeledpeptide identifies the compound as an agonist and displacement oflabeled peptide identifies the compound as an antagonist. In oneembodiment, the proximity of the two labels is measured usingradioactive or fluorescent probes. In another embodiment, the ROR LBD islabeled with glutathione-S-transferase (GST), and the peptide is labeledwith biotin. In another embodiment, wherein the peptide has the sequenceERRTVLQLLLGNSNK (SEQ ID NO: 3), wherein biotin is linked to theN-terminus of the peptide by an aminohexanoic acid linker.

The present invention also provides a method of increasing the number ofT cells reactive to a specific antigen, comprising administering an RORγagonist in conjunction with, or subsequent to, administration of theantigen.

The present invention also provides a method of increasing theimmunogenicity of an immunogenic composition in an individual in needthereof, comprising administering an immunogenicity-increasing amount ofan RORγ agonist in conjunction with, or subsequent to, the immunogeniccomposition. In one embodiment, the immunogenic composition is a vaccinecomposition. In another embodiment, the vaccine composition is anattenuated live vaccine or a non-replicating and/or subunit vaccine,wherein the vaccine induces memory T_(H)-17 cells specific for saidvaccine. In one embodiment, the vaccine is a tumor vaccine, viralvaccine, bacterial vaccine or parasitic vaccine. In one embodiment, theviral vaccine is a DNA viral vaccine, an RNA viral vaccine or aretroviral viral vaccine.

The present invention also provides a method of increasing mucosalimmunity to a preselected antigen, comprising administering to a subjecta mucosal immunity-increasing amount of an RORγ agonist in conjunctionwith, or subsequent, to the antigen. In one embodiment, the antigen is abacterial antigen, viral antigen or tumor antigen.

The present invention also provides a method of enhancing induction,expression and/or release of a pro-inflammatory cytokine, apro-inflammatory cytokine receptor, a pro-inflammatory chemokine or apro-inflammatory chemokine receptor in cells capable of expressing saidcytokine, cytokine receptor, chemokine or chemokine receptor, comprisingadministering an RORγ agonist to the cells. In one embodiment, the RORγagonist is a small organic molecule, protein, peptide, nucleic acid,carbohydrate or antibody. In another embodiment, the pro-inflammatorycytokine is IL-17 or IL-22. In one embodiment, the cells are contactedin vitro, ex vivo or in vivo.

The present invention also provides a method of inducing T_(H)-17 celldifferentiation and/or transcription of IL-17 and/or IL-22 in apopulation of T cells that may include T_(H)-17 cells, comprisingcontacting the cells with an effective amount of an RORγ agonist. In oneembodiment, the cells are contacted in vitro, ex vivo or in vivo.

The present invention also provides a method of inducing T_(H)-17 celldifferentiation in an individual in need thereof, comprisingadministering an effective T_(H)-17 cell differentiation-inducing amountof an RORγ agonist to the individual

The present invention also provides a method of inhibiting T_(H)-17 celldifferentiation and/or release of IL-17 and/or IL-22, comprisingadministering an effective amount of an RORγ antagonist to a populationof T cells that may include T_(H)-17 cells. In one embodiment, theantagonist is a small organic molecule, protein, peptide, nucleic acid,carbohydrate or antibody. In another embodiment, the T cell is a CD4+ Tcell, a CD8+ T-cell or a TCRγδ+ T cell. In one embodiment, theantagonist is administered in vitro, ex vivo or in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Naive T cell differentiation in mouse. T_(H)-17 celldifferentiation is independent of T_(H)1 and T_(H)2. IFNγ and IL-4, fromT_(H)1 and T_(H)2, respectively, antagonize T_(H)-17 differentiation,while TGFβ and IL-6 stimulate T_(H)-17 differentiation. RORγt isrequired for T_(H)-17 cell formation, and its expression appears to takeplace early in differentiation (Ivanov, McKenzie et al. 2006). IL-23appears to have a stimulatory effect on the differentiated T_(H)-17cell.

FIG. 2. Nuclear Receptor Structure. The activation function-1 (AF-1)domain induces gene transcription independently of ligand. Theactivation function-2 domain (AF-2) is a part of the LBD and is requiredfor the ligand-dependent transcriptional effect.

FIG. 3. Dose response of RORγ antagonists in a Chinese Hamster Ovary(CHO) cell assay of transcription. CHO cells were transfected with anexpression plasmid encoding a fusion of the DNA-binding domain (DBD) ofthe yeast transcriptional factor Gal4 with the ligand-binding domain ofmouse RORγ (Gal4-mRORγ). A luciferase reporter containing a 5×Gal4response element at its promoter region was cotransfected with thereceptor chimera. T0901317 (Schultz, Tu et al. 2000) and OR-1050 areadded in DMSO and each point is the median of triplicate values,normalized to a DMSO-only control.

FIGS. 4A-B. Identification and characterization of RORγ agonists. Tolower the transcriptional basal activity of RORγ in CHO cells andincrease the dynamic range for RORγ activation by agonists, onemicromolar of the RORγ antagonist T0901317 (Cayman Chemical, Ann ArborMich.) was added to the cell-based assay described in FIG. 3. (FIG. 4A)OR-942 (hyodeoxycholic acid methyl ester) was identified as an agonistin the transcriptional assay. When OR-942 was tested alone, GAL4-RORγtranscriptional activity was only modestly elevated in CHO cells. In thepresence of the antagonist T0901317, however, RORγ displays loweredbasal activity and OR-942 activates RORγ transcriptional activity byapproximately 8-fold.

(FIG. 4B) The agonist activity of OR-942 was confirmed in a biochemicalassay based on coregulatory peptide recruitment. OR-942 induces aninteraction between partially purified RORγ LBD, expressed as a fusionprotein with glutathione-S-transferase (GST-RORγ), and a 15-mer peptide(ERRTVLQLLLGNPTK; SEQ ID NO: 4), or peptide K, derived from the humancoregulator protein RIP140 (Lee, Elwood et al. 2002). The peptide isbiotinylated at the amino-terminal. The interaction of peptide K andGST-RORγ was followed by fluorescence resonance energy transfer (FRET).The two components of the assay were labeled with an anti-GST antibodycoupled to allophycocyanin (APC) and streptavidin-R-phycoerythrin(SA-RPE). FRET units were calculated as the fractional increase (×100)of the APC/RPE fluorescence ratio over the value obtained in thepresence of a mutated form of the K peptide (Kmut, ERRTVLQLVVGNPTK; SEQID NO: 5), also biotinylated at the N-terminal amino acid residue, thatsubstitutes two of the leucine residues required for coactivator peptidebinding (Darimont, Wagner et al. 1998; Li, Lambert et al. 2003) withvaline.

FIG. 5. The RORγ antagonist T0901317 has the properties of a competitiveantagonist to OR-942. A dose response of the RORγ agonist OR-942 wascarried out in the biochemical assay with GST-RORγ and peptide K. Theeffect of the antagonist T0901317 is to shift the estimated EC₅₀ ofOR-942 from 0.4 μM to 4 μM, consistent with competition for a singlebinding site on RORγ. Each point is the median of triplicate values.

FIG. 6. Simultaneous characterization of an RORγ agonist and antagonistin the biochemical assay for RORγ. The activities of OR-1050 andOR-12872 were compared in a coregulatory peptide recruitment assay thatuses peptide biotinylated K1 (from RIP140 of rat) instead of peptide K(from RIP140 of human). The sequence of peptide K1 is ERRTVLQLLLGNSNK(SEQ ID NO: 3). The mutated form of the K1 peptide (K1mut,ERRTVLQLVVGNSNK; SEQ ID NO: 6) was used as a control. The biotin wasseparated from the K1 and K1mut N-terminus by an aminohexanoic acidlinker. The combination of GST-RORγ and peptide K1 has a significantFRET value in the absence of added ligand, and this enablescharacterization of both agonist and antagonist in the same assayformat. Values are the average of duplicate measurements.

FIG. 7. Regulation of T_(H)-17 cell differentiation by RORγ ligands.Naïve CD4⁺CD62L⁺ T cells were incubated for five days in the presence ofIL-6 and TGFβ and 0.01% DMSO. On day 5, a high level of intracellularcytokine expression is induced by combination of treatment with aphorbol ester, ionomycin, and brefeldin A, and T_(H)-17 and T_(H) cellsidentified by intracellular staining with antibodies to IL-17 and IFN-γ,respectively. The percentage of IL-17 positive cells, or T_(H)-17 cells,was calculated as a fraction of total live cells. OR-1050 decreases theproportion of IL-17⁺ cells while OR-12872 increases the fraction ofIL-17⁺ cells. Each treatment was performed in duplicate (mean±SD, n=2)

FIGS. 8A-D. An RORγ agonist (OR-12872) reverses antagonist (OR-1050)effects on regulation of T_(H)-17 cell differentiation. The methodsfollow FIG. 7. Each treatment was performed in duplicate, and arepresentative scatterplot for each treatment is shown. FIG. 8A:control; FIG. 8B: 3 μm OR-1050; FIG. 8C: 3 μm OR-12872; FIG. 8D: 3 μmOR-1050+3 μm OR-12872.

FIG. 9A-D. OR-885 inhibits T_(H)-17 differentiation and OR-12872reverses its antagonistic effect. The methods follow FIGS. 7 and 8. Eachtreatment was performed in duplicate, and a representative scatterplotfor each treatment is shown. FIG. 9A: control; FIG. 9B: 3 μm OR-885;FIG. 8C: 1 μm OR-112872; FIG. 8D: 3 μm OR-885+1 μm OR-12872.

FIG. 10. IL-17 release into culture medium was measured four days afterinduction of T_(H)-17 differentiation in CD4⁺ naïve murine T cells,following the methods of FIG. 7. Each antagonist (OR-885, OR-1050,OR-13571, and OR-2161) was incubated in the cultures at 3 μM, in thepresence or absence of OR-12872, also at 3 μM. On day 4, culturesupernatants were saved and IL-17 measured by ELISA.

FIGS. 11A-C. RORγ antagonist inhibits severity, weight loss, onset,Th-17 frequency and inflammation in EAE models. (FIGS. 11A-C) C57BL/6mice were immunized with 150 μg MOG₃₅₋₅₅ at day 0 to induce EAE and weredosed daily with corn oil (vehicle) or OR-1050 (50 mg/kg, 2× per day) byoral gavage starting at day −1. At Day 26, the spinal cord was removedfor analysis of inflammation (FIG. 11C).

FIGS. 11D-F SJL/J mice were immunized with 75 μg PLP₁₃₉₋₁₅₁ (Day 0).Osmotic pumps delivering vehicle or OR-1050 (˜30 mg/kg) were inserted atDays 3 and 4. Day of onset (FIG. 11E) compares the first day when EAEseverity is 1 or higher. The ratio of Th-17 cells (CD4⁺CD8⁻IL17⁺) tototal live cells was analyzed in a separate study (t FIG. 11F) at day 9after immunization with PLP where mice had been dosed daily with OR-1050(100 mg/kg in HRC-6) since day −1. Statistics (panels A,B,D,F) wereperformed by Student's t-test. *, P<0.05; #, P<0.01 (mean±sem, n=7-10)or by the Mann-Whitney rank order test (FIGS. 11C, E, median valuesshown).

FIG. 11G shows the effect of OR-1050 on splenic T_(H)-17 cells inC57BL/6 mice (29% inhibition, p<0.05, n=8-10) with no change in T_(H)1cells. C57BL/6 female mice were immunized with 150 μg MOG33-55 on studyday 0. Mice were treated with either OR-1050 at 100 mg/kg or withvehicle (HRC-6) by oral gavage for 9 days starting day 0. Splenocyteswere collected at Day 9 and stained for T_(H)-17 (CD4⁺CD8⁻IL17⁺) andT_(H)1 (CD4⁺CD8⁻IFNγ) cells. Statistics were performed by Student'st-test.

FIG. 12A. OR-1050 inhibits the onset of paralysis in a murine model ofEAE. This is a separate statistical analysis of data presented in FIG.11A. C57BL/6 mice were immunized with MOG₃₅₋₅₅, an immunogenic peptidederived from myelin oligodendrocyte glycoprotein, to trigger symptoms ofEAE. Mice were treated with corn oil vehicle or OR-1050 (15 mg/kg or 50mg/kg) twice per day by gavage starting one day before injection ofMOG₃₅₋₅₅ and continuing for 25 days after injection. The day on which avisual, clinical score first reached 1 or greater was recorded for eachmouse, and observations were carried out until day 26. By a rank ordernon-parametric test for multiple groups (Kruskal-Wallis, with Dunn'sMultiple Comparison Test for significance), the onset of EAE (determinedas a severity score of 1 or greater) in mice treated with 100 mg/kgOR-1050 per day is significantly (p<0.05) delayed compared to thevehicle control. A similar finding was obtained by analysis of day ofonset determined by severity score of 2 or greater.

FIG. 12B. Bioavailability of OR-1050. OR-1050 was dosed in CD-1 mice at5 mg/ml in HRC-6, a proprietary formulation that is commerciallyavailable from Pharmatek (San Diego, Calif.). Blood samples fromtriplicate animals were tested for levels of OR-1050 at multiple timepoints.

FIGS. 13A-F. IL-17 levels in lymph node cells from mice immunized withmyelin-derived peptides respond to cognate peptide antigens, IL-23 andRORγ ligands. (FIG. 13A) SJL/J mice were immunized by complete Freund'sadjuvant (CFA) only or by Proteolipid Protein residues 139-151(PLP₁₃₉₋₁₅₁) emulsified in CFA. Lymph nodes were cultured for 3 days inwith or without 40 μg/ml PLP in the presence or absence of OR-885.Culture media were analyzed by ELISA for IL-17. (FIGS. 13B-C) Lymph nodecells from PLP-immunized SJL/J mice were cultured as before with theaddition of 10 ng/ml IL-23 and 3 μM compounds. At the end of 1 day or 3days in culture, Th-17 (CD4⁺CD8⁻IL17⁺) cell frequency was measured byFACS (FIG. 13B) and compared to IL-17 levels in culture media (FIG.13C). (FIG. 13D-F) Lymph node cells from MOG₃₅₋₅₅ immunized C57BL/6 micewere cultured for 3 days with 30 μg/ml MOG and/or 10 ng/ml IL-23. RORγligands were not added (FIG. 13D), or added at the beginning of theculture at a single concentration (FIG. 13E, 1 μM) or in multiple doses(FIG. 13F).

FIG. 14. Inhibition of IL-17 release in mouse lymph node cultures by theRORγ antagonist OR-885 can be reversed by the RORγ agonist OR-12872. Thelymph node cells were culture in the presence of 10 ng/mL IL-23 and 40μg/mL PLP for three days under identical conditions to FIG. 13 and IL-17in the culture supernatant was measured by ELISA.

FIGS. 15A-B. IL-22 release in lymph node cultures is regulated by RORγligands. Lymph node cells from MOG-immunized C57BL/6 mice were culturedfor 3 days with 30 μg/ml MOG, 10 mg/ml IL-23 (FIG. 15A) and 3 μMcompounds (FIG. 15B). Culture media were analyzed by IL-22 ELISA.

FIGS. 16A-C. Human IL-17 release from activated human T cells isinhibited by RORγ antagonist and is partially reversible in the presenceof OR-12872, an RORγ agonist. In FIG. 16A, PBMCs were exposed to 1 μg/mlCon A for 3 days and treated simultaneously with 3 μM OR-885 or DMSOcarrier in the presence or absence of 3 μM OR-12872. In FIG. 16B, PBMCswere treated for 3 days in the presence 1 μg/mL Con A and 3 M of thefollowing compounds: OR-13571, OR-1050, or OR-12872 alone or incombination. In FIG. 16C, T cell blasts were treated with OR-1050,OR-13571, and OR-12872 in the presence of a phorbol ester for 18 hoursto induce cytokine release.

FIGS. 17A-B. RORγ antagonist regulation of CIA in DBA/1 mice. Mice wereimmunized with bovine type II collagen (CII) at days 0 and 29. (FIG.17A) Arthritic scores in hind limb (max score=8) for vehicle-treated(n=15) or 100 mg/kg/day OR-1050-treated (n=12) animals. (FIG. 17B)Disease intensity (Mean±SEM) is the area under the curve (AUC) for hindlimb clinical scores from day 24 to 48. AUC scores for the two groupswere compared by the Mann-Whitney rank order test (one-tailed) todetermine statistical significance.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention includes the discovery that small moleculeantagonists to retinoic acid related orphan receptor γ (RORγ) inhibitT_(H)-17 cell differentiation from naïve mouse T cells in cell culture,and inhibit the release of cytokines from mouse and human T_(H)-17cells. These small molecule antagonists have unique and beneficialproperties for treatment of inflammatory and autoimmune diseases, sincethey inhibit the release of several pro-inflammatory cytokines fromT_(H)-17 cells, including IL-17 and IL-22. Conversely, a potent RORγagonist can enhance T_(H)-17 cell formation and reverse the antagonisteffect. These small molecule inhibitors can be used to treat a varietyof inflammatory and autoimmune disorders including, but not limited to,arthritis, diabetes, multiple sclerosis, uveitis, rheumatoid arthritis,psoriasis, asthma, bronchitis, allergic rhinitis, chronic obstructivepulmonary disease, atherosclerosis, H. pylori infections, ulcersresulting from H. pylori infections and inflammatory bowel disease(IBD). IBD includes, but is not limited to, Crohn's disease, ulcerativecolitis and sprue.

RORγ agonists and antagonists are fairly specific to T_(H)-17 cells anddo not appear to have a primary effect on T_(H)1 cells. Severalstrategies that inhibit development or activity of T_(H)-17 cells forthe treatment of MS and other autoimmune diseases can be envisioned, butthese primarily involve treatment with macromolecules such as antibodiesthat block IL-23 or IL-17 (Bowman, Chackerian et al. 2006) or cytokinesthat inhibit T_(H)-17 cell function (Batten, Li et al. 2006; Stumhofer,Laurence et al. 2006). RORγ antagonists offer the first approach tospecifically block T_(H)-17 development with small molecule drugs. AnRORγ antagonist offers superior properties for human dosing andtherapeutic use such as oral bioavailability. The following definitionsare provided:

A receptor antagonist is a molecule that inhibits the normalphysiological function of a receptor. Many drugs work by blocking theaction of endogenous receptor agonists such as hormones andneurotransmitters. Antagonists that compete with an agonist for areceptor are competitive antagonists. Those that antagonize by othermeans are non-competitive antagonists.

A receptor agonist is a molecule that, in the nuclear receptor context,increases the transcriptional activity of a receptor or overcomes theactivity of a competitive receptor antagonist.

A small molecule drug is an orally or intravenously bioavailablecompound having a molecular weight less than about 600 daltons.

Related nuclear receptor. The human and mouse families of nuclearreceptors comprise 48 members (Laudet 1999), and these have beenarranged in both families (e.g., family 1, including both RORγ and LXRα)and subfamilies (e.g., NR1F) that include RORα, RORβ, and RORγ. For thepurposes of this discussion, related nuclear receptors are those thatbelong to a single receptor subfamily, such as NR1F, and unrelatednuclear receptors may belong to the same family but to differentsubfamilies.

A confirmed hit at RORγ is a small molecule compound that regulatestranscription through RORγ and also regulates the affinity of the RORγLBD with an appropriate coregulatory peptide in a validated assay forthe receptor.

Potency. A measure of compound concentration required to activate orinhibit a pharmacological endpoint. Potency is often estimated as EC₅₀(effective concentration producing a 50% effect) or by similar measuresknown to those skilled in the art of pharmacology.

Rank order of potency is a method of comparing small molecule ligandpharmacology data from two separate assays. Compounds are ranked by EC₅₀or other measure of potency in each of the two assays. The purpose ofthis ranking is to provide a method of comparing the results of twodifferent assays for which absolute compound potencies may differ.

A transcriptional assay for a nuclear receptor measures regulation ofgene expression of a target gene that contains a response element forthat receptor in its promoter region. Promoter regions can be engineeredto contain appropriate response elements and these in turn can becoupled to a variety of reporter genes. Such assays are widely used tocharacterize nuclear receptor function and utilize target genes, such aschloramphenicol acetyl transferase (CAT), luciferase (LUC), andbeta-lactamase (BLA). The activity of these enzymes is readily assayedin cell extracts or whole cells. Nuclear receptor assays also takeadvantage of the fact that the ligand-binding domain (LBD) of thereceptor can function independently of its DNA-binding domain (DBD).Chimeric receptors that contain a common DBD, for example the DBD of theyeast transcriptional factor GAL4 (Webster, Green et al. 1988), arefused to the LBDs of individual nuclear receptors. Multiple nuclearreceptor LBDs, fused to the same DBD, may be screened against a commontarget gene that contains the response element for that DBD (Schultz, Tuet al. 2000). Chimeric receptors are co-transfected individually withthe common target gene. Ligands effects are determined after incubationfor a sufficient period of time according to changes in level ofexpression of the target gene.

A biochemical assay for a nuclear receptor is carried out in thepresence of partially purified receptor LBD and, directly or indirectly,measures binding of ligand. Two methods are commonly used: (i)competitive displacement of known, labeled ligand and (ii) recruitmentof coregulatory peptide. Coregulatory peptide recruitment takesadvantage of the fact that the receptor LBD will usually bind shortpeptides derived from conserved sequences within the so-calledcoregulatory proteins in a manner that depends on the presence orabsence of ligand (Bramlett, Yao et al. 2000; Lee, Elwood et al. 2002;Wu, Chin et al. 2002). Coregulatory proteins in turn are required fortranscriptional regulation in response to ligand binding to the receptor(McKenna and O'Malley 2002). Both receptor LBD and peptide are taggedwith molecular markers, and the degree of association of these markersdetermined in the presence or absence of ligand.

RORγ.

The gene that encodes RORγ (RORc) undergoes alternative splicing to giverise to two splicing isoforms, RORγ and RORγt. The expression of RORγ iswidespread, and mRNA for RORγ appears in liver, muscle, fat and manyother tissues (Jetten, Kurebayashi et al. 2001). The RORγt splicingisoform is predicted to generate a protein that is truncated by 21 aminoacids at the N-terminus of RORγ (He, Deftos et al. 1998). RORγt isexpressed predominantly in thymocytes, lymph node precursors known asLTi cells, in T_(H)-17 cells, and in other T cells, including asubpopulation of CD8⁺, γδTCR⁺, and NKT cells (Ivanov, McKenzie et al.2006; Ivanov and Littman. 2007). The RORγt isoform contains an LBD thatis identical to RORγ. For the purpose of this application, the tworeceptors may be referred to collectively as RORγ.

RORγ Function.

Nuclear receptors are thought to act primarily as nuclear transcriptionfactors, by regulating gene expression. Some nuclear receptors have beenshown to have acute, non-genomic effects that cannot be explained bygene transcription. Examples include the thyroid and estrogen receptors(Hiroi, Kim et al. 2006). Although a transcriptional assay is useful foridentification of RORγ ligands, the ligand response of RORγ may includedirect activation of other cell-signaling pathways, such as thosemediated by kinases, phosphatases, or levels of intracellularmessengers.

FITC—Flourescein isothiocyanate

APC—allophycocyanin

PE—phycoerythrin

LBD—ligand-binding domain

DBD—DNA-binding domain

FRET—Fluorescence resonance energy transfer

The T_(H)-17 cell is recently-described subset of CD4⁺ T helper cellsthat expresses IL-17 and IL-17F, as well as other cytokines, includingpro-inflammatory cytokines such as IL-22 (Liang, Tan et al. 2006). TheT_(H)-17 cell also expresses the autocrine cytokine, IL-21. Other Tcells may also express RORγt and IL-17. The most prevalent type ofIL-17⁺ T cell in the gut is the TCRαβ⁺CD4⁺ T_(H)-17 cell (Ivanov,McKenzie et al. 2006), while the γδ T cell is the primary source ofIL-17 in response to pulmonary infection (Lockhart, Green et al. 2006).In addition, CD8⁺ T cells, γδ T cells and NKT cells also express RORγ.Thus, T_(H)-17 cells are a subset of IL-17⁺ T cells. A significantfraction of the IL-17+ T cells appear to be RORγt⁺ (Ivanov and Littman2007).

Pharmaceutical composition refers to a mixture of RORγ antagonist orinhibitor, or a pharmaceutically acceptable salt, prodrug, derivative ormetabolite thereof, with other chemical components, such as diluents orcarriers. The pharmaceutical composition facilitates administration ofthe compound to an organism. Multiple techniques of administering acompound exist in the art including, but not limited to, oral,injection, aerosol, parenteral, and topical administration.Pharmaceutical compositions can also be obtained by reacting compoundswith inorganic or organic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike.

Carrier defines a chemical compound that facilitates the incorporationof a compound into cells or tissues. For example dimethyl sulfoxide(DMSO) is a commonly utilized carrier as it facilitates the uptake ofmany organic compounds into the cells or tissues of an organism.

Diluent defines chemical compounds diluted in water that will dissolveor suspend the compound of interest and preferably also stabilize thebiologically active form of the compound. Salts dissolved in bufferedsolutions are utilized as diluents in the art. One commonly usedbuffered solution is phosphate buffered saline because it mimics thesalt conditions of human blood. Since buffer salts can control the pH ofa solution at low concentrations, a buffered diluent rarely modifies thebiological activity of a compound.

Physiologically acceptable defines a carrier or diluent that is suitablefor in vivo administration.

Pharmaceutically acceptable salt refers to a salt form of an originalcompound formed by association of a counterion with that compound,wherein the counterion is generally nontoxic. Pharmaceutical salts canbe obtained, for example, by reacting a compound of the invention withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonicacid, p-toluenesulfonic acid, salicylic acid and the like.Pharmaceutical salts can also be obtained by reacting a compound of theinvention with a base to form a salt such as an ammonium salt, an alkalimetal salt, such as a sodium or a potassium salt, an alkaline earthmetal salt, such as a calcium or a magnesium salt, a salt of organicbases such as dicyclohexylamine, N-methyl-D-glutamine,tris(hydroxymethyl)methylamine, and salts with amino acids such asarginine, lysine, and the like.

Metabolite refers to a compound to which a RORγ antagonist or agonist isconverted within the cells of a mammal. The pharmaceutical compositionsof the present invention may include a metabolite of a RORγ antagonistinstead of the RORγ antagonist. The scope of the methods of the presentinvention includes those instances where the RORγ antagonist isadministered to the patient, yet the metabolite is the bioactive entity.

Prodrug refers to an agent that is converted into the parent drug invivo. Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug. An example, without limitation, of a prodrug wouldbe a compound of the present invention which is administered as an ester(the “prodrug”) to facilitate transmittal across a cell membrane wherewater solubility is detrimental to mobility but which then ismetabolically hydrolyzed to the carboxylic acid, the active entity, onceinside the cell where water-solubility is beneficial. A further exampleof a prodrug might be a short peptide (polyaminoacid) bonded to an acidgroup where the peptide is metabolized to reveal the active moiety.

In a further aspect, the present invention relates to a method oftreating a patient with a pharmaceutical composition as describedherein.

The term “treating” or “treatment” does not necessarily mean total cure.Any alleviation of any undesired signs or symptoms of the disease to anyextent or the slowing down of the progress of the disease can beconsidered treatment. Furthermore, treatment may include acts that mayworsen the patient's overall feeling of well being or appearance.Treatment may also include lengthening the life of the patient, even ifthe symptoms are not alleviated, the disease conditions are notameliorated, or the patient's overall feeling of well being is notimproved.

RORγ Antagonists

The present invention provides compounds that selectively inhibit thetranscriptional activity of the orphan nuclear receptor RORγ. These RORγantagonists may be small organic molecules, proteins, peptides, nucleicacids, carbohydrates or antibodies. These compounds permit theproperties of RORγ as a pharmacological target to be investigated inisolated cells and in animals. Further, the invention provides for novelmethods of treating autoimmune disease and related conditions byinhibiting the function and/or activity of T_(H)-17 cells, or byinhibiting the differentiation of T_(H)-17 cells, or by inhibiting IL-17and IL-22 release in a population of T cells that may include T_(H)-17cells (e.g., CD4+ T cells) and other RORγt+IL-17+ T cells such as CD8+ Tcells, NKT, or TCRγδ+ T cells, through suppression of the activity ofRORγ. In examples shown herein, specific compounds that inhibit oractivate transcription through the RORγ LBD, respectively RORγantagonists or agonists, are described, and structurally distinctantagonists of RORγ transcription specifically inhibit thedifferentiation of CD4⁺ T_(H)-17 cells from naïve CD4⁺ T cells.Furthermore, the structurally distinct antagonists also inhibit therelease of IL-17 from murine T_(H)-17 cells during differentiation orfrom memory IL-17⁺ T cells in lymph node cultures. In addition, thestructurally diverse RORγ antagonists inhibit IL-17 release from humanperipheral blood mononuclear cells (PBMCs).

The examples demonstrate that pharmacologically useful small moleculesfor cellular studies can be identified by receptor assays. To be useful,the molecules should be active in an assay of receptor-mediatedtranscription. Second, in addition to activity in a transcriptionalassay, such molecules should be active in a second receptor assay thatuses a mechanistically distinct readout, such as coactivator orcoregulatory protein or peptide recruitment to receptor LBD as afunction of ligand concentration (Heery, Kalkhoven et al. 1997; Lee,Elwood et al. 2002). The proximity of the RORγ LBD and the coregulatorypeptide or macromolecule is measured by one of several well-establishedbiophysical methods. Some of these methods, well known to practitionersof the art of studying protein-peptide interactions, includefluorescence resonance energy transfer (FRET), fluorescencepolarization, time-resolved FRET (TR-FRET) with europium conjugates asdonor, and Alpha Screen, in which laser-induced oxygen emission from adonor bead stimulates a fluorophore in an acceptor bead in closeproximity (Iannone, Consler et al. 2001; Lee, Elwood et al. 2002; Xu,Stanley et al. 2002; Moore, Galicia et al. 2004; Li, Choi et al. 2005).

The biochemical assay excludes molecules that non-specifically regulatean RORγ reporter gene in culture. To allow definitive findings in cellexperiments, a compound will ideally have an EC₅₀<1 μM and measurablecytotoxicity only at higher concentrations. Finally, such moleculesshould be selective for RORγ. If this is not the case, a secondarycontrol should be available, for example, an agonist that demonstratesthe reversibility of antagonist effect. Without these controls, there isthe opportunity to confuse an effect of a non-specific candidate RORγligand on T_(H)-17 cells with an authentic activity of the smallmolecule mediated by interaction with the LBD of RORγ. Thetranscriptional and biochemical assays described herein can be used todetermine the ability of any small molecule to act as an RORγ receptorantagonist or agonist.

The invention provides examples in which the validity of certain hitswere further confirmed by demonstrating that a series of relatedcompounds to the hit share a similar rank order of potency in both thetranscriptional and biochemical assays over a wide range.

In view of the fact that RORγt is required for CD4⁺ T_(H)-17 cellformation in mice (Littman and Eberl 2006), we tested whether RORγligands specifically inhibit T_(H)-17 differentiation from naïve CD4⁺ Tcells in culture (Mangan, Harrington et al. 2006; Veldhoen, Hocking etal. 2006). T_(H)-17 inhibition by small molecule modulation through RORγwas verified by several criteria. First, three separate structurallydistinct RORγ antagonists (OR-1050, OR-885, and OR-13571, EC₅₀<1 μM inthe transcriptional assay) had comparable activity in T_(H)-17inhibition; second, antagonist effect could be reversed by a potent andspecific agonist (OR-12872) to RORγ; and third, the RORγ ligand effectwas specific for cell culture differentiation of T_(H)-17 but notT_(H)-1 cells.

Further, the invention provides for molecules with a reasonable marginof safety. We demonstrated that one of the RORγ ligands, OR-1050, can bedosed in mice such that it should cause a pharmacological effect throughRORγ. We therefore examined the effect of the compound on function ofthe thymus, since the major pool of developing T lymphocytes, the doublepositive CD4⁺CD8⁺ (DP) T cells, expresses RORγt. Germline deletion ofRORγ or RORγt leads to an increased rate of DP thymocyte apoptosis and areduction in DP thymocyte number to about 30% of control (Kurebayashi,Ueda et al. 2000; Sun, Unutmaz et al. 2000; Eberl and Littman 2004).Therefore, a model study with OR-1050 was used to investigate possibleeffects on thymic function. The RORγ antagonist OR-1050 also activatesthe nuclear receptors LXRα and LXRβ, and therefore the bioavailabilityof OR-1050 could be demonstrated by induction of triglycerideaccumulation in liver since this is a well-defined endpoint for LXRligands (Schultz, Tu et al. 2000; Beyer, Schmidt et al. 2004). The EC₅₀for OR-1050 at LXRα (1.5 M) is higher than for RORγ (0.3 M) intranscriptional assays, suggesting that RORγ would also be antagonizedin the drug-treated animals. In a six day study, liver triglycerideswere markedly elevated by daily exposure to 100 mg/kg OR-1050, but thecompound had no effect on the frequency or total number of the majorcell types of the thymus. This invention therefore provides forcompounds that will have limited effects on the function of the thymus.

Further, OR-1050 slows the onset of EAE in C57BL/6 female mice injectedwith a myelin-derived peptide. The data are consistent with theprediction that an RORγ antagonist free of LXR activity will beeffective in this model. In addition, OR-1050 is demonstrated to havemodest bioavailability. The invention provides a method for selectingcompounds that have negligible or reduced LXR potency while maintainingor improving RORγ potency. T0901317 does not elevate liver triglyceridelevels in mice where the LXRα and LXRβ receptors have been deleted(Schultz, Tu et al. 2000). More specific RORγ antagonists are predictedto have beneficial effects in animal models of CIA, EAE, and IBD whilelimiting the undesirable consequences of LXR activation, such as livertriglyceride accumulation and elevated serum LDL (Schultz, Tu et al.2000; Beyer, Schmidt et al. 2004; Groot, Pearce et al. 2005), or otherundesirable consequences, either for safety or efficacy, of activation,inhibition, or interaction with other known pharmacological targets.

In addition, the invention provides for compounds that suppress IL-17expression in activated human T cells. Elevated IL-17 levels in diseasetissue are strongly suggested to signal pathogenic involvement ofT_(H)-17 and other IL-17 positive cells.

The advantages of direct targeting of T_(H)-17 cells with a smallmolecule drug could be very significant compared to the biologics thatare very likely under development (Bowman, Chackerian et al. 2006). Inone embodiment, a drug from the RORγ antagonist class has good oralbioavailability. In another embodiment, the drug has a half-life of fourhours or more, enabling once or twice-daily administration. Nuclearreceptor ligands have commonly given rise to orally bioavailable drugs;OR-1050 is an example of an orally-bioavailable RORγ antagonistcharacterized in these studies. The advantage of an orally bioavailabledrug is: (i) direct oral administration is feasible; (ii) unlikeinjectable biologics, which may have an extremely long half life, asmall molecule drug with reasonable half-life can be withdrawn ifnecessary to limit side effects and (iii) small molecule drugs arereadily manufactured. Such compounds may also be selected for clinicaldevelopment by potency in animal models of EAE, CIA and IBD and byparallel in vivo studies of compound safety.

RORγ Agonists

RORγ agonists may be small organic molecules, proteins, peptides,nucleic acids, carbohydrates or antibodies. Particularly preferred aresmall organic molecules. The present invention also includes methods ofincreasing the number of T cells reactive to a specific antigen byadministering an RORγ agonist in conjunction with, or subsequent to,administration of the antigen; and for enhancing the differentiation ofT_(H)-17 cells by contacting a population of T cells that may includeT_(H)-17 cells with an RORγ agonist. In one embodiment, agonists of RORγare used to enhance or modulate an immune response. In anotherembodiment, an RORγ agonist is used to enhance the effectiveness ofvaccine compositions. Vaccination remains the primary mechanism ofinhibiting the spread of infectious agents. For newly discoveredorganisms, vaccination is the most highly favored option since drugtherapy may require decades to provide clinical options. Although mosthealthy individuals respond to the foreign antigen presented byvaccination, there is a critical need to stimulate the immune responsein the elderly, who are much less responsive to vaccination in the firstplace due to aging of their immune system. Furthermore, subunitvaccines, although easy to prepare since they are based on exogenouslyexpressed proteins from the pathogen, are often not highly immunogenic.Thus, concomitant stimulation of the immune response will improvevaccine take. The vaccine compositions may be attenuated live vaccines,or non-replicating and/or subunit vaccines. In one embodiment, thesevaccines induce memory T_(H)-17 cells specific for the vaccine. Types ofvaccines include, but are not limited to, tumor vaccines, viral vaccines(DNA, RNA or retroviral), bacterial vaccines and parasitic vaccines.

RORγ agonists can also be used to increase mucosal immunity to apreselected antigen by administering a mucosal immunity-enhancing amountof an RORγ agonist in conjunction with, or subsequent to, the antigen.The antigen can be bacterial, viral or a tumor antigen.

An RORγ agonist can be used to increase the immunogenicity of animmunogenic composition (e.g., vaccine composition) by administering theRORγ agonist in conjunction with, or subsequent to, the immunogeniccomposition. RORγ agonists can also be used to enhance induction,expression and/or release of a pro-inflammatory cytokine (e.g., IL-6,IL-17, IL-22, IL-3, TNF-α), a pro-inflammatory cytokine receptor, apro-inflammatory chemokine (e.g., CC chemokines, CXC chemokines, Cchemokines, CX₃C chemokines) or a pro-inflammatory chemokine receptor incells capable of expressing the cytokine, cytokine receptor, chemokineor chemokine receptor, by administering the RORγ agonist to the cells.The administration may be in vitro, ex vivo or in vivo.

The RORγ agonists described herein also induce T_(H)-17 celldifferentiation and/or transcription of IL-17 and/or IL-22 in apopulation of T cells that may include T_(H)-17 cells. The RORγ agonistsmay be administered in vitro, ex vivo or in vivo.

The pharmaceutical compositions described herein can be administered toa human patient per se, or in pharmaceutical compositions where they aremixed with other active ingredients, as in combination therapy, orsuitable carriers or excipient(s). Techniques for formulation andadministration of the compounds of the instant application may be foundin “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton,Pa., 18th edition, 1990. The pharmaceutical compositions may also beadministered to other mammals, including dogs, cats, sheep, pigs,horses, cows, and the like. Thus, the veterinary use of thesepharmaceutical compositions is also within the scope of the presentinvention.

Suitable routes of administration may, for example, include oral,rectal, topical, transmucosal, or intestinal administration; parenteraldelivery, including intramuscular, subcutaneous, intravenous,intramedullary injections, as well as intrathecal, directintraventricular, intraperitoneal, intranasal, or intraocularinjections.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directly inthe renal or cardiac area, often in a depot or sustained releaseformulation. Furthermore, one may administer the drug in a targeted drugdelivery system, for example, in a liposome coated with atissue-specific antibody. The liposomes will be targeted to and taken upselectively by the organ.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or tabletting processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. Any of the well-knowntechniques, carriers, and excipients may be used as suitable and asunderstood in the art; e.g., in Remington's Pharmaceutical Sciences,above.

For injection, the RORγ agonists/antagonists of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by mixing one or more solid excipient withpharmaceutical combination of the invention, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

For topical administration, the compounds may be formulated foradministration to the epidermis as ointments, gels, creams, pastes,salves, gels, creams or lotions, or as a transdermal patch. Ointmentsand creams may, for example, be formulated with an aqueous or oily basewith the addition of suitable thickening and/or gelling agents. Lotionsmay be formulated with an aqueous or oily base and will in general alsocontaining one or more emulsifying agents, stabilizing agents,dispersing agents, suspending agents, thickening agents, or coloringagents.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally, includingsublingually, which include push-fit capsules made of gelatin, as wellas soft, sealed capsules made of gelatin and a plasticizer, such asglycerol or sorbitol. The push-fit capsules can contain the activeingredients in admixture with filler such as lactose, binders such asstarches, and/or lubricants such as talc or magnesium stearate and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycols. In addition, stabilizers maybe added. All formulations for oral administration should be in dosagessuitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. A commoncosolvent system used is the VPD co-solvent system, which is a solutionof 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate80™, and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. Naturally, the proportions of a co-solvent system may be variedconsiderably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of POLYSORBATE 80™; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, thecompounds may be delivered using a sustained-release system, such assemipermeable matrices of solid hydrophobic polymers containing thetherapeutic agent. Various sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

Many of the compounds used in the pharmaceutical combinations of theinvention may be provided as salts with pharmaceutically compatiblecounterions. Pharmaceutically compatible salts may be formed with manyacids, including but not limited to hydrochloric, sulfuric, acetic,lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble inaqueous or other protonic solvents than are the corresponding free acidor base forms.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions where the active ingredients are contained in anamount effective to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount of compound effectiveto prevent, alleviate or ameliorate symptoms of disease or prolong thesurvival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art, especially in light of the detailed disclosureprovided herein.

The exact formulation, route of administration and dosage for thepharmaceutical compositions of the present invention can be chosen bythe individual physician in view of the patient's condition. (See e.g.,Fingl et al. 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1p. 1). Typically, the dose range of the composition administered to thepatient can be from about 0.5 to 1000 mg/kg of the patient's bodyweight. The dosage may be a single one or a series of two or more givenin the course of one or more days, as is needed by the patient. Notethat for almost all of the specific compounds mentioned in the presentdisclosure, human dosages for treatment of at least some condition havebeen established. Thus, in most instances, the present invention willuse those same dosages, or dosages that are between about 0.1% and 500%,more preferably between about 25% and 250% of the established humandosage. Where no human dosage is established, as will be the case fornewly-discovered pharmaceutical compounds, a suitable human dosage canbe inferred from ED₅₀ or ID₅₀ values, or other appropriate valuesderived from in vitro or in vivo studies, as qualified by toxicitystudies and efficacy studies in animals.

Although the exact dosage will be determined on a drug-by-drug basis, inmost cases, some generalizations regarding the dosage can be made. Thedaily dosage regimen for an adult human patient may be, for example, anoral dose of between 0.1 mg and 6000 mg of each ingredient, preferablybetween 1 mg and 5000 mg, e.g. 25 to 5000 mg or an intravenous,subcutaneous, or intramuscular dose of each ingredient between 0.01 mgand 100 mg, preferably between 0.1 mg and 60 mg, e.g. 1 to 40 mg of eachingredient of the pharmaceutical compositions of the present inventionor a pharmaceutically acceptable salt thereof calculated as the freebase, the composition being administered 1 to 4 times per day.Alternatively the compositions of the invention may be administered bycontinuous intravenous infusion, preferably at a dose of each ingredientup to 400 mg per day. Thus, the total daily dosage by oraladministration of each ingredient will typically be in the range 1 to2500 mg and the total daily dosage by parenteral administration willtypically be in the range 0.1 to 400 mg. Suitably the compounds will beadministered for a period of continuous therapy, for example for a weekor more, or for months or years.

In one embodiment, the dose of the pharmaceutical composition comprisinga RORγ agonist or antagonist or a pharmaceutically acceptable salt,prodrug, derivative or metabolite thereof, is from about 10 to about 50mg per day.

In another embodiment, the RORγ antagonist is administered daily withoutresulting in weight loss or hypertriglyceridemia.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themodulating effects, or minimal effective concentration (MEC). The MECwill vary for each compound but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC assays orbioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compositionsshould be administered using a regimen that maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. The pack or dispensermay also be accompanied with a notice associated with the container inform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby the agency of the form of the drug for human or veterinaryadministration. Such notice, for example, may be the labeling approvedby the U.S. Food and Drug Administration for prescription drugs, or theapproved product insert. Compositions comprising a compound of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

The compositions described herein may also be used in the preparation ofa medicament for treatment of any of the disorders described above.

Example 1

This example describes ligands to RORγ and confirms their receptorregulation properties in two mechanistically distinct receptor assays.Transcriptional assays for mouse and human RORγ (mRORγ and hRORγ) wereimplemented in cultured cells. A biochemical assay, using exogenouslyexpressed mRORγ LBD, was established to measure ligand-mediatedregulation of coregulatory peptide recruitment in vitro. Thetranscriptional assay measures the product of a reporter gene, such asan enzyme, whose expression is in turn regulated by the RORγ LBD. Thebiochemical assay responds to a ligand-dependent conformational changein the receptor LBD that changes receptor affinity for coregulatorypeptide. The ligands to RORγ described here were predominantlyidentified in the transcriptional assay and confirmed in the biochemicalassay.

This example also describes several additional tests that were commonlyperformed to confirm the authenticity of a hit to RORγ. These tests alsoverified the validity of the transcriptional and biochemical assaysused. In all cases, the activity of a compound was also tested intranscriptional assays for the orphan nuclear receptors RORα, RORβ, andSF-1 to demonstrate specificity and to rule out a non-specific effect onthe assay system as an explanation for the apparent activity. In somecases, close analogs of a confirmed hit were obtained by synthesis orpurchase from a commercial source. The series of compounds related tothe confirmed hit was then tested in the biochemical and transcriptionalassays. The rank order of the potency of the compounds was thencompared. If closely similar for the two assays, the findingsdemonstrated that minor chemical modifications had equivalent effects oncompound interaction with the RORγ LBD in each assay. The data imply inturn that the compounds are binding to the same binding pocket. Eventhough absolute EC₅₀ values may differ in a rank order comparison, theobservation that relative values are similar provides strongconfirmation of the conclusion that the identified compounds actdirectly through RORγ and not by a secondary mechanism that is assay butnot receptor-specific. This results presented in this exampledemonstrate that the transcriptional and biochemical assays provide aninternally consistent method of characterizing RORγ ligand potency andthat more potent compounds can be identified using this assay technologyby a program of additional screening or directed medicinal chemistrysynthesis.

Methods

Transcriptional Assay.

Gal4-mRORγ was generated by inserting the LBD of mouse RORγ(Ile-251-Lys-516) into the EcoRI-HindIII site of pFA-CMV. The vectorpFA-CMV (Stratagene, La Jolla, Calif.) contains the yeast Gal4 DNAbinding domain (amino acids 1-148) upstream of the multiple cloning sitewhere the EcoRI and HindIII restriction enzyme sites are found. Thefragment of mRORγ was a PCR product using the template of pM-mRORγ(Medvedev, Yan et al. 1996). The complete Gal4-mRORγ nucleotide sequenceis shown below:

(Gal4 DBD) (SEQ ID NO: 7)ATGAAGCTACTGTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAGTGCTCCAAAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTACTCTCCCAAAACCAAAAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAATCAAGGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGAGAAGACCTTGACATGATTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAACAGGATTATTTGTACAAGATAATGTGAATAAAGATGCCGTCACAGATAGATTGGCTTCAGTGGAGACTGATATGCCTCTAACATTGAGACAGCATAGAATAAGTGCGACATCATCATCGGAAGAGAGTAGTAACAAAGGTCAAAGACAGTTGACTGTATCGCCG  (linker) (SEQ ID NO: 8)GGATCCGCCCGGGCTGGAATTCGC (mRORγ LBD) (SEQ ID NO: 9)ATTCCCAGTTTCTGCAGTGCCCCAGAGGTACCATATGCCTCTCTGACAGACATAGAGTACCTGGTACAGAATGTCTGCAAGTCCTTCCGAGAGACATGCCAGCTGCGACTGGAGGACCTTCTACGGCAGCGCACCAACCTCTTTTCACGGGAGGAGGTGACCAGCTACCAGAGGAAGTCAATGTGGGAGATGTGGGAGCGCTGTGCCCACCACCTCACTGAGGCCATTCAGTATGTGGTGGAGTTTGCCAAGCGGCTTTCAGGCTTCATGGAGCTCTGCCAGAATGACCAGATCATACTACTGACAGCAGGAGCAATGGAAGTCGTCCTAGTCAGAATGTGCAGGGCCTACAATGCCAACAACCACACAGTCTTTTTTGAAGGCAAATACGGTGGTGTGGAGCTGTTTCGAGCCTTGGGCTGCAGCGAGCTCATCAGCTCCATATTTGACTTTTCCCACTTCCTCAGCGCCCTGTGTTTTTCTGAGGATGAGATTGCCCTCTACACGGCCCTGGTTCTCATCAATGCCAACCGTCCTGGGCTCCAAGAGAAGAGGAGAGTGGAACATCTGCAATACAATTTGGAACTGGCTTTCCATCATCATCTCTGCAAGACTCATCGACAAGGCCTCCTAGCCAAGCTGCCACCCAAAGGAAAACTCCGGAGCCTGTGCAGCCAACATGTGGAAAAGCTGCAGATCTTCCAGCACCTCCACCCCATCGTGGTCCAAGCCGCCTTCCCNCCACTCTATAAGGAACTCTTCAGCACTGATGTTGAATCCCCTGAGGGGCTGTCAAAGTG A The complete Gal4-mROR protein sequence is shown below:

(Gal4 DBD) (SEQ ID NO: 10)MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSP (linker)(SEQ ID NO: 11) GSARAGIR (mRORγ LBD) (SEQ ID NO: 12)IPSFCSAPEVPYASLTDIEYLVQNVCKSFRETCQLRLEDLLRQRTNLFSREEVTSYQRKSMWEMWERCAHHLTEAIQYVVEFAKRLSGFMELCQNDQIILLTAGAMEVVLVRMCRAYNANNHTVFFEGKYGGVELFRALGCSELISSIFDFSHFLSALCFSEDEIALYTALVLINANRPGLQEKRRVEHLQYNLELAFHHHLCKTHRQGLLAKLPPKGKLRSLCSQHVEKLQIFQHLHPIVVQAAFXPLY KELFSTDVESPEGLSKGal4-hRORγ was generated by inserting the ligand-binding domain of humanRORγ LBD (Ser-253-Lys-518) into pFA-CMV by BamHI-KpnI sites. The vectorpFA-CMV (Stratagene, La Jolla, Calif.) contains the yeast Gal4 DNAbinding domain (amino acids 1-148) at N-terminal of the multiple cloningsites. The fragment of hRORγ LBD was a PCR product using the template ofa commercial clone from Invitrogen (Clone ID: 5186655; Vector:pCMV-SPORT6). The complete Gal4-hRORγ nucleotide sequence is shownbelow:

(Gal4 DBD) (SEQ ID NO: 7)ATGAAGCTACTGTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAGTGCTCCAAAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTACTCTCCCAAAACCAAAAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAATCAAGGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGAGAAGACCTTGACATGATTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAACAGGATTATTTGTACAAGATAATGTGAATAAAGATGCCGTCACAGATAGATTGGCTTCAGTGGAGACTGATATGCCTCTAACATTGAGACAGCATAGAATAAGTGCGACATCATCATCGGAAGAGAGTAGTAACAAAGGTCAAAGACAGTTGACTGTATCGCCG  (linker) (SEQ ID NO: 13)GGATCC (hRORγ LBD) (SEQ ID NO: 14)AGCCCCAGTTTCCGCAGCACACCGGAGGCACCCTATGCCTCCCTGACAGAGATAGAGCACCTGGTGCAGAGCGTCTGCAAGTCCTACAGGGAGACATGCCAGCGGCTGGAGGACCTGCTGCGGCAGCGCTCCAACATCTTCTCCCGGGAGGAAGTGACTGGCTACCAGAGGAAGTCCATGTGGGAGATGTGGGAACGGTGTGCCCACCACCTCACCGAGGCCATTCAGTACGTGGTGGAGTTCGCCAAGAGGCTCTCAGGCTTTATGGAGCTCTGCCAGAATGACCAGATTGTGCTTCTCAAAGCAGGAGCAATGGAAGTGGTGCTGGTTAGGATGTGCCGGGCCTACAATGCTGACAACCGCACGGTCTTTTTTGAAGGCAAATACGGTGGCATGGAGCTGTTCCGAGCCTTGGGCTGCAGCGAGCTCATCAGCTCCATCTTTGACTTCTCCCACTCCCTAAGTGCCTTGCACTTTTCCGAGGATGAGATTGCCCTCTACACAGCCCTTGTTCTCATCAATGCCCATCGGCCAGGGCTCCAAGAGAAAAGGAAAGTAGAACAGCTGCAGTACAATCTGGAGCTGGCCTTTCATCATCATCTCTGCAAGACTCATCGCCAAAGCATCCTGGCAAAGCTGCCACCCAAGGGGAAGCTTCGGAGCCTGTGTAGCCAGCATGTGGAAAGGCTGCAGATCTTCCAGCACCTCCACCCCATCGTGGTCCAAGCCGCTTTCCCTCCACTCTACAAGGAGCTCTTCAGCACTGAAACCGAGTCACCTGTGGGGCTGTCCAAGTGAThe complete Gal4-hRORγ protein sequence is shown below:

(Gal4 DBD) (SEQ ID NO: 10)MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSP (linker)(SEQ ID NO: 15) GS  (hRORγ LBD) (SEQ ID NO: 16)SPSFRSTPEAPYASLTEIEHLVQSVCKSYRETCQLRLEDLLRQRSNIFSREEVTGYQRKSMWEMWERCAHHLTEAIQYVVEFAKRLSGFMELCQNDQIVLLKAGAMEVVLVRMCRAYNADNRTVFFEGKYGGMELFRALGCSELISSIFDFSHSLSALHFSEDEIALYTALVLINAHRPGLQEKRKVEQLQYNLELAFHHHLCKTHRQSILAKLPPKGKLRSLCSQHVERLQIFQHLHPIVVQAAFPPLY KELFSTETESPVGLSK

Four other orphan nuclear receptors were similarly cloned as Gal4-LBDhybrids in pFA-CMV by PCR as follows for initial screening studies:human SF-1 (NR5A1, aa 198-462) at BamHI and XbaI restriction enzymessites; mRORα (NR1F1, aa 266-523) at XmaI-HindIII restriction enzymessites; human RORβ (NR1F2, aa 201-459) at EcoRI and KpnI restrictionenzyme sites; and mouse LRH-1 (NR5A2, aa 311-561) at EcoRI and HindIIIrestriction enzyme sites.

A firefly luciferase reporter gene for Gal4 DBD hybrid receptors, suchas Gal4-RORγ, contains five copies of the Gal4 17-mer response elementin its promoter and the coding sequence for firefly luciferase (pG5-luc,Promega). CHO (Chinese Hamster Ovary) cells were cultured in Ham's F12medium supplemented with 10% fetal calf serum (Gemini Biologicals), 10μg/ml penicillin and streptomycin, and were maintained in a humidified37° C. incubator (Thermo Electron, Steri-Cycle) with 5% CO₂. 24 hoursbefore transfection, cells were plated in T175 flasks (5×10⁶cells/flask) or T75 flasks (1.7×10⁶ cells/flask). Transient transfectionof CHO cells was performed using TransiT-CHO reagents (Mirus, Madison,Wis.) with a mixing ratio of 2:6:1 between plasmid DNA (in Gtg),TransIT-CHO (in μl) and CHO-mojo (in μl). As an example, 9 μg pG5-luc,8.75 μg pcDNA3 and 0.25 μg receptor plasmid were transfected into eachT175 flask.

Four hours after transfection, cells were dislodged with trypsin andseeded into a 384-well plate at 8,000 cells/well using a TitertekMultidrop 384. Edge effects within the 384-well plate were minimized byleaving plates at room temperature for 30 min so that cells platedevenly within each well (Lundholt, Scudder et al. 2003). Plates aremaintained at 37° C. in a humidified atmosphere with 7% CO₂ overnight.Four (4) hours after cell seeding, compounds dissolved in DMSO at 10 mMwere added to cells at final concentrations of 20 micromolar (μM) to 20nanomolar (nM) in 0.2% DMSO in triplicate plates. Approximately 40 hourslater, cytotoxicity in the assay well was characterized by addition ofthe dye resazurin (O'Brien, Wilson et al. 2000) to a final concentrationof 3 M. After incubation for 2 hours at 37 degrees centigrade, theconversion of resazurin to resorufin was measured by fluorescence(excitation at 570 nm and emission at 615 nm). Living cells catalyze dyeconversion while cells that lack metabolic activity do not. Percentviability was determined as 2 hour fluorescence, minus background at t=0hours, normalized to DMSO controls. For detection of luciferaseactivity, media was removed from plates after the measurement ofresazurin conversion, and SteadyLite luciferase reagent (Perkin-Elmer)was added (30 l/well). Luminescence is detected with a Victor 2-V platereader (Perkin-Elmer) and normalized to luciferase activity of DMSOcontrol wells alone.

Compounds Described.

The following test compounds were obtained commercially: T0901317(Cayman Chemicals); rockogenin or OR-885 (Steraloids, Newport, R.I.);5α, 20α, 22α, 25D-Spirostan-3β, 12β-diol-11-one or OR-345 (Steraloids);hyodeoxycholic acid methyl ester or OR-942 and hyodeoxyclolic acid orOR-412 (Steraloids); 11-oxo ursolic acid acetate or OR-13571(Microsource, Gaylordsville, Conn.) and AG-205/33159060 (Specs, Delft,The Netherlands) or OR-2161; OR-133008 (Specs AG-690/40752395);OR-133097 (Specs AG-690/40698971); OR-133099 (Specs AG-690/40699006);OR-133167 (Specs AG-690/40752726); and OR-133171 (SpecsAG-690/40752859).

Compounds Synthesized.

The following describes synthesis of a representative antagonist (e.g.,OR-1050) and a representative agonist (e.g., OR-12872) to RORγ. Allstarting materials were commercially available. Related antagonists andagonists, where not purchased commercially, were synthesized byanalogous methods.

Abbreviations: Ac₂O—acetic anhydride; NEt₃—triethylamine;THF—tetrahydrofuran; DMAP—4-Dimethylaminopyridine;EDCl—1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride;TBSCl—tert-butyl-chloro-dimethyl-silane; DMF—dimethylformamide;TBAF—Tetra-n-butylammonium fluoride.

Synthetic Scheme for OR-1050 where R=Para-Nitrophenyl:

2-(4-Ethylamino-phenyl)-1,1,1,3,3,3-hexafluoro-propan-2-ol

To a solution of 3.0 gm of2-(4-Amino-phenyl)-1,1,1,3,3,3-hexafluoro-propan-2-ol (MatrixScientific, Columbia, S.C.) in 30 ml of methylene chloride was added 2.4ml of triethyl amine, 1.2 ml of acetic anhydride, and 100 mg ofdimethylaminopyridine. The mixture was allowed to stir at roomtemperature for 16 hrs. An additional 2.4 ml of triethyl amine and 1.2ml of acetic anhydride was added and the mixture was stirred for anadditional 6 hrs. The solution was evaporated and purified by columnchromatography (1:1 Ethyl Acetate:Hexane) to give 3.1 gm of materialthat was used in the next step. To this material dissolved in 30 ml oftetrahydrofuran cooled to 0 degrees C. was added 1.6 gm of lithiumaluminum hydride. This solution was stirred for 16 hrs and quenched with1 N sodium hydroxide. This was then extracted with ethyl acetate anddried over anhydrous sodium sulfate. Chromatography gave 2.188 gm of thenamed product. 1H NMR was consistent with the structure.

N-Ethyl-4-nitro-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide(OR-1050)

To a solution of 104 mg2-(4-Ethylamino-phenyl)-1,1,1,3,3,3-hexafluoro-propan-2-ol in 3 ml ofpyridine was added 104 mg 4-Nitro-benzenesulfonyl chloride and 10 mg ofdimethylaminopyridine. The mixture was stirred for 16 hrs and thenconcentrated in vacuo. After chromatography (1:3 Ethyl Acetate:Hexane),129 mg of the named product was obtained. 1H NMR was consistent with thestructure.

Synthetic scheme for OR-12872

4R-[3R,6R-Bis-(tert-butyl-dimethyl-silanyloxy)-10R,13R-dimethyl-5R-8S-9S-14S-hexadecahydro-cyclopenta[a]phenanthren-17R-yl]-pentanoicacid methyl ester (2)

To a solution of 8.974 gm of hyodeoxycholic acid methyl ester(4R-(3R,6R-Dihydroxy-10 R,13R-dimethyl-5R-8S-9S-14S-hexadecahydro-cyclopenta[a]phenanthren-17R-yl)-pentanoicacid methyl ester) (1) (Steraloids, Newport, R.I.) in 60 ml of dimethylformamide was added 12 ml of triethylamine, 8.3 gm oftert-Butyl-chloro-dimethyl-silane and 270 mg of dimethyl-aminopyridine.The mixture was stirred at room temperature for 16 hrs and concentratedin vacuo. After chromatography (1:9 Ethyl Acetate:Hexane), 13.748 gm ofthe named product (2) was obtained. 1H NMR was consistent with thestructure.

4R-[3R,6R-Bis-(tert-butyl-dimethyl-silanyloxy)-10R,13R-dimethyl-5R-8S-9S-14Shexadecahydro-cyclopenta[a]phenanthren-17R-yl]-pentanoic acid (3)

To a solution of4-[3,6-Bis-(tert-butyl-dimethyl-silanyloxy)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-17-yl]-pentanoicacid methyl ester (9.41 gm) in 15 ml of tetrahydrofuran, 10 ml ofmethanol and 10 ml of water was added 620 mg of sodium hydroxide. Themixture was then stirred at room temperature for 16 hrs and concentratedin vacuo. After acidification the residue was extracted with ethylacetate, dried over anhydrous sodium sulfate, filtered and concentratedin vacuo. Chromatography (1:3 Ethyl Acetate:Hexane) gave 8.6 gm of thetitle compound (3). 1H NMR was consistent with the structure.

5R-[3R,6R-Bis-(tert-butyl-dimethyl-silanyloxy)-10R,13R-dimethyl-5R-8S-9S-14S-hexadecahydro-cyclopenta[a]phenanthren-17R-yl]-hexan-2-one(4)

To a solution of4-[3,6-Bis-(tert-butyl-dimethyl-silanyloxy)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-17-yl]-pentanoicacid (3.419 gm) in 30 ml of tetrahydrofuran at 0 degrees C. was added7.6 ml of a 1.6 M solution of methyl lithium. The mixture was stirred at0 degrees C. for 30 min and quenched with 20 ml water followed by 20 mlof 1N hydrochloric acid. The mixture was extracted with ethyl acetate,dried over anhydrous sodium sulfate and concentrated in vacuo.Chromatography (1:9 Ethyl Acetate:Hexane) gave 0.985 gm of the titlecompound (4). 1H NMR was consistent with the structure.

5R-[3R,6R-Bis-(tert-butyl-dimethyl-silanyloxy)-10R,13R-dimethyl-5R-8S-9S-14S-hexadecahydro-cyclopenta[a]phenanthren-17R-yl]-hexan-2-ol(5)

To a solution of 516 mg of5-[3,6-Bis-(tert-butyl-dimethyl-silanyloxy)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-17-yl]-hexan-2-onein 10 ml of ethanol was added 250 mg of sodium borohydride. The mixturewas stirred for 1 hr and concentrated in vacuo, 20 ml of water was addedfollowed by 30 ml of ethyl acetate and 10 ml of 1N hydrochloric acid.The mixture was extracted with ethyl acetate, dried over anhydroussodium sulfate and concentrated in vacuo. Chromatography (1:9 EthylAcetate:Hexane) gave 512 mg of the title compound (5). 1H NMR wasconsistent with the structure.

17R-(4-Methoxy-1R-methyl-pentyl)-10R,13R-dimethyl-5R-8S-9S-14S-hexadecahydro-cyclopenta[a]phenanthrene-3R,6R-diol(OR-12872)

To a solution of 262 mg of (5) in 6 ml of dimethyl formamide was added110 ml of methyl iodide followed by 29 mg of a 60% oil dispersion ofsodium hydride. The mixture was stirred for 16 hr and concentrated invacuo. The mixture was extracted with ethyl acetate, dried overanhydrous sodium sulfate and concentrated in vacuo. Chromatography (1:9Ethyl Acetate:Hexane) gave 257 mg of an intermediate that was dissolvedin 10 ml of tetrahydrofuran and 2.4 ml of a IM tetrahydrofuran solutionof tetra-butylammonium fluoride. The mixture was then stirred at roomtemperature for 16 hrs and then at 60 C for 2 hrs. After concentrationin vacuo and chromatography (1:9 Ethyl Acetate:Hexane), 154 mg of thetitle compound (OR-12872) was produced. 1H NMR was consistent with thestructure.

Compound Nomenclature.

Compounds have the following IUPAC names: OR-1048,4-Bromo-N-ethyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide;OR-1052,N-Ethyl-4-methyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide;OR-1050,N-Ethyl-4-nitro-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide;OR-1047,4-Butyl-N-ethyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide;OR-1031,N-Ethyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide;T0901317,N-(2,2,2-Trifluoro-ethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide;OR-1030,N-Ethyl-N-[4-(2,2,2-trifluoro-1-methoxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide;OR-1046,{Benzenesulfonyl-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-amino}-aceticacid.

OR-12872,17-(4-Methoxy-1-methyl-pentyl)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthrene-3,6-diol;OR-12866,17-(4-Methoxy-1,4-dimethyl-pentyl)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthrene-3,6-diol;OR-942,4-(3,6-Dihydroxy-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-17-yl)-pentanoicacid methyl ester; OR-12863,17-(4-Hydroxy-1-methyl-butyl)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthrene-3,6-diol;OR-12870,5-(3,6-Dihydroxy-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-17-yl)-hexan-2-one;OR-12868,4-(3,6-Dihydroxy-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-17-yl)-pentanoicacid dimethylamide; OR-12864,17-(4-Methoxy-1-methyl-butyl)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthrene-3,6-diol;OR-12865,17-(4-Hydroxy-1,4-dimethyl-pentyl)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthrene-3,6-diol;OR-12871,17-(4-Hydroxy-1-methyl-pentyl)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthrene-3,6-diol;OR-412,4-(3,6-Dihydroxy-hexadecahydro-cyclopenta[a]phenanthren-17-yl)-pentanoicacid; OR-12867,4-(3,6-Dihydroxy-hexadecahydro-cyclopenta[a]phenanthren-17-yl)-pentanoicacid methylamide; OR-12869,4-(3,6-Dihydroxy-hexadecahydro-cyclopenta[a]phenanthren-17-yl)-1-piperidin-1-yl-pentan-1-one;

OR-13571 (11-oxo-ursolic acid acetate),10-Acetoxy-1,2,6a,6b,9,9,12a-heptamethyl-13-oxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydro-2H-picene-4a-carboxylicacid.

The following descriptions are available for two of the naturalcompounds tested: OR-885 (CAS registry number, 16653-52-4, Rockogenin,5α, 20α, 22α, 25D-SPIROSTAN-3β, 12β-DIOL, Steraloids catalogue numberS0300-000) and OR-345 (5α, 20α, 22α, 25D-SPIROSTAN-3β, 12β-DIOL-11-ONE,Steraloids catalogue number S0400-000).

OR-2161 isN-(4,6-Di-piperidin-1-yl-[1,3,5]triazin-2-yl)-N′-(2-methoxy-3-nitro-benzylidene)-hydrazine(Specs catalog number AG-205/33159060); OR-133008 is2-{5-[(4-Chloro-phenylamino)-methyl]-4-ethyl-4H-[1,2,4]triazol-3-ylsulfanyl}-N-naphthalen-1-yl-acetamide(Specs AG-690/40752395); OR-133097 is2-{5-[(3-Chloro-phenylamino)-methyl]-4-ethyl-4H-[1,2,4]triazol-3-ylsulfanyl}-N-naphthalen-1-yl-acetamide(Specs AG-690/40698971); OR-133099 is2-{4-Ethyl-5-[(3-trifluoromethyl-phenylamino)-methyl]-4H-[1,2,4]triazol-3-ylsulfanyl}-N-naphthalen-1-yl-acetamide(Specs AG-690/40699006); OR-133167 is2-{5-[(3-Chloro-phenylamino)-methyl]-4-methyl-4H-[1,2,4]triazol-3-ylsulfanyl}-N-naphthalen-1-yl-acetamide(Specs AG-690/40752726); OR-133171 is2-{5-[(3-Chloro-4-methyl-phenylamino)-methyl]-4-ethyl-4H-[1,2,4]triazol-3-ylsulfanyl}-N-naphthalen-1-yl-acetamide(Specs AG-690/40752859).

Biochemical Assay.

GST-RORγ was generated by subcloning the RORγ-LBD into pET-41a, a vectorwhich expresses GST fusion proteins in bacteria. A biochemical assaydeveloped with GST-RORγ purified from bacteria had a poor response toligand. The receptor was expressed in a baculovirus expression vector(Luckow and Summers 1989) in insect Sf9 cells. Expression in Sf9 cells,as an alternative to bacterial expression, was also successfully used togenerate RORα LBD in a form suitable for protein crystallization(Kallen, Schlaeppi et al. 2002).

The cloning steps for GST-RORγ were as follows. The mRORγ LBD wasexcised from Gal4-mRORγ (see above for cloning steps) at BamHI andHindIII sites of Sequence 1 and cloned into the corresponding sites ofpET-41a(+) (EMD Biosciences, San Diego, Calif.). A GST-mRORγ fragmentwas then excised from pET-41a(+) at XbaI and XhoI sites and cloned intoa pcDNA3.1vector (Invitrogen, Carlsbad, Calif.) restricted at NheI andXhoI sites. Plasmid GST-mRORγ-pcDNA3.1 was used as a PCR template togenerate a GST-mRORγ fragment that was later cloned into pENTR/D-TOPOvector (Invitrogen, Carlsbad, Calif.) by standard cloning techniquesrecommended by the manufacturer of the vector. The nucleotide sequenceof GST-mRORγ is shown below:

(GST) (SEQ ID NO: 17) ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCC AAAATCGGAT  (linker)(SEQ ID NO: 18) GGTTCAACTAGTGGTTCTGGTCATCACCATCACCATCACTCCGCGGGTCTGGTGCCACGCGGTAGTACTGCAATTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACCTGGACAGCCCAGATCTGGGTACCGGTGGTGGCTCCGGTGATGACGACGACAAGAGTCCCATGGGATATCGGGGATCCGCCCG GGCTGGAATTCGC (mRORγLBD) (SEQ ID NO: 9) ATTCCCAGTTTCTGCAGTGCCCCAGAGGTACCATATGCCTCTCTGACAGACATAGAGTACCTGGTACAGAATGTCTGCAAGTCCTTCCGAGAGACATGCCAGCTGCGACTGGAGGACCTTCTACGGCAGCGCACCAACCTCTTTTCACGGGAGGAGGTGACCAGCTACCAGAGGAAGTCAATGTGGGAGATGTGGGAGCGCTGTGCCCACCACCTCACTGAGGCCATTCAGTATGTGGTGGAGTTTGCCAAGCGGCTTTCAGGCTTCATGGAGCTCTGCCAGAATGACCAGATCATACTACTGACAGCAGGAGCAATGGAAGTCGTCCTAGTCAGAATGTGCAGGGCCTACAATGCCAACAACCACACAGTCTTTTTTGAAGGCAAATACGGTGGTGTGGAGCTGTTTCGAGCCTTGGGCTGCAGCGAGCTCATCAGCTCCATATTTGACTTTTCCCACTTCCTCAGCGCCCTGTGTTTTTCTGAGGATGAGATTGCCCTCTACACGGCCCTGGTTCTCATCAATGCCAACCGTCCTGGGCTCCAAGAGAAGAGGAGAGTGGAACATCTGCAATACAATTTGGAACTGGCTTTCCATCATCATCTCTGCAAGACTCATCGACAAGGCCTCCTAGCCAAGCTGCCACCCAAAGGAAAACTCCGGAGCCTGTGCAGCCAACATGTGGAAAAGCTGCAGATCTTCCAGCACCTCCACCCCATCGTGGTCCAAGCCGCCTTCCCNCCACTCTATAAGGAACTCTTCAGCACTGATGTTGAATCCCCTGAGGGGCTGTCAAAGTG AThe protein sequence of GST-mRORγ is shown below:

(Gal4 DBD) (SEQ ID NO: 10)MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSP (linker)(SEQ ID NO: 11) GSARAGIR (mRORγ LBD) (SEQ ID NO: 12)IPSFCSAPEVPYASLTDIEYLVQNVCKSFRETCQLRLEDLLRQRTNLFSREEVTSYQRKSMWEMWERCAHHLTEAIQYVVEFAKRLSGFMELCQNDQIILLTAGAMEVVLVRMCRAYNANNHTVFFEGKYGGVELFRALGCSELISSIFDFSHFLSALCFSEDEIALYTALVLINANRPGLQEKRRVEHLQYNLELAFHHHLCKTHRQGLLAKLPPKGKLRSLCSQHVEKLQIFQHLHPIVVQAAFXPLY KELFSTDVESPEGLSK

For baculoviral expression of RORγ, the cDNA fragment of GST-RORγ-LBD,was subcloned from pET-41a into the pENTR/D-TOPO vector using acommercially-available kit (BaculoDirect, Invitrogen). Briefly, GST-RORγcarried by pENTR/D-TOPO was transfected into mono-layer Sf9 cells(8×10⁵/well in 6-well plates) by Cellfectin (Invitrogen). Sf9 cells wereincubated at 27° C. in a non-humidified incubator for 72 hours. Cellmedia was collected as P1 viral stock and used to further infect Sf9cells to generate P2 and P3 virus. Significant signs of infection wereobserved during generation of P3 virus. A plaque assay was performed todetermine the titer of P3 virus and to isolate single plaques. P4 viruswas generated from a single plaque and propagated on a larger scale.

Adherent SF9 cells were infected with baculovirus carrying GST-RORγ andwere cultured for 72 hours. Sf9 cell were then subject to a briefsonication in a lysis buffer (20 mM phosphate, pH 6.8, 50 mM KCl, 50 mMNaCl, 0.5 mM EDTA, 50% glycerol, 0.001% NP-40 and 1 mM DTT) andcentrifuged at 3000 rpm for 15 minutes. GST-RORγ protein represents anestimated 20-30% of total soluble protein in the cytosolic supernatantas determined by SDS-PAGE and immunoblot with anti-GST antibody (Sigma).Some protein remained in an insoluble fraction after centrifugation. Wefound that addition of RORγ ligands (preferably 5 μM T0901317) in theculture media increased the fraction of GST-RORγ in the cytosolicfraction. Cleared cell lysate was stored at −70° C. and is directlyutilized in the coregulatory peptide recruitment assay.

To carry out the biochemical assay, receptor, peptides, andfluorescently-labeled probes to GST and biotin were incubated underthese conditions: purified biotinylated peptide K (0.2 μM), 80 nManti-GST coupled to allophycocyanin (APC), 25 nMstreptavidin-R-phycoerythrin (SA-RPE), that specifically binds thebiotinylated peptide, 20 mM sodium phosphate, pH 6.8, 50 mM KCl, 50 mMNaCl, 0.5 mM EDTA, 5% glycerol, 0.001% NP-40, and 1 mM DTT wereincubated with the Sf9 cell lysate diluted in the range of 1:10 to 1:50,following methods from the literature (Coward, Lee et al. 2001; Drake,Zhang et al. 2002; Lee, Elwood et al. 2002), and incubated at roomtemperature for 2 hours. The labeled probes anti-GST-APC and SA-RPE werepurchased from Prozyme (San Leandro, Calif.). Compounds were addeddirectly from a DMSO solution to a final concentration of 1-2% DMSO.Fluorescence was measured on a Wallac Victor 2 V plate reader equippedwith a 670/40 nm filter (Omega Optical, Brattleboro, Vt.) for APCemission (Channel A) and a 600/25 bandpass filter for monitoring the RPEemission (Channel B). The FRET signal is calculated as: (the differencebetween the ratio of Channel B/Channel A in the presence of an activepeptide (such as peptide K) and the Channel A/Channel B ratio in thepresence of the corresponding inactive peptide (peptide Kmut))×100 or inthe complete absence of peptide.

Results

Members of the RORα, RORβ, and RORγ family of orphan nuclear receptorsare generally recognized as having a high basal activity in cell culturesystems (Jetten, Kurebayashi et al. 2001). The transcriptional activityof Gal4-mRORγ in CHO cells and in the choriocarcinoma cell line JEG-3was >20-fold higher than a mutated form of Gal4-mRORγ (E502Q or E/Q).The E/Q mutation inactivates a highly conserved residue of the helix 12region of the LBD that is required for recruitment of transcriptionalcoactivators in most well-characterized nuclear receptors (Li, Lambertet al. 2003), and is predicted to abrogate transcription from the RORγLBD. An assay for detecting RORγ ligands was developed in CHO cells, andabout 100,000 individual small molecule compounds from variouscommercial sources tested in this assay in several formats (e.g., in 96or 384-well plates, or following 24-hour or 40-hour incubation with testcompounds). Two classes of RORγ antagonists are shown below. Structure 1represents a genus of non-steroidal antagonists in which R1=H, C1-C6Alkyl, F, Cl, Br, I, NO2; R2=C1-C4 Alkyl; and X═OH. Structure 3represents a genus of steroidal antagonists in which X═O or is absent(X═H,H). Any antagonist encompassed by these generic structures iswithin the scope of the present invention. The ability of any suchcompound, or any other compound, to act as an RORγ antagonist may beeasily determined using the assays described herein. Structure 2represents a genus of steroidal agonists in which X═C(E)(F)(G); whereinE and F are independently selected from H, lower alkyl or E and F takentogether form a carbonyl group; G is selected from OH, O-lower alkyl orN(lower alkyl)₂. Any agonist encompassed by these generic structures iswithin the scope of the present invention. The ability of any suchcompound, or any other compound, to act as an RORγ agonist may be easilydetermined using the assays described herein.

FIG. 3 shows that two compounds, T0901317 and OR-1050 (Table 1), bothinhibit the high basal transcriptional activity of RORγ. In separatestudies, we determined that the two compounds had no effects on thetranscription of RORα, RORβ or SF-1 as GAL4 hybrids in the same formatas the GAL4-RORγ assay. Further, T0901317<10 μM was not cytotoxic to CHOcells while OR-1050 was not cytotoxic at 50 μM. All antagonists andagonists identified in this example were similarly specific for RORγ inthe transcriptional assay and were non-cytotoxic to CHO cells at 20 μMor, if not, were non-cytotoxic at a concentration 10-fold greater thanthe EC₅₀ in the transcriptional assay.

Because the basal transactivation activity of GST-RORγ is high, othercompounds that bind RORγ and have only a modest effect on transcriptionwhen tested alone in cell culture may be overlooked. Some of thesecompounds could be classified as RORγ agonists. These may activate,rather than repress, RORγ-mediated processes in cells or animals.Accordingly, a screening assay for RORγ agonists was carried out inwhich one of the RORγ antagonists identified, T0901317, was added tosuppress the basal transcriptional activity of GAL4-RORγ. FIG. 4Ademonstrates a method for the identification of RORγ agonists. Compoundsthat only modestly elevated transcription in the absence of T0901317(for example, the steroidal antagonist OR-942, see Table 2), but induceda much greater elevation in the presence of 1-3 μM T0901317, determinedas the ratio between wells containing OR-942 and those lacking OR-942,were further investigated in dose response assays (FIG. 4A).

To further confirm the properties of RORγ agonists and antagonists, abiochemical assay for RORγ was developed. This assay can be used toscreen for agonists or antagonists of RORα, RORγ or RORβ3. This assay isbased on contacting a compound with a labeled, expressed ROR LBD and apeptide that includes residues 710-720 (RTVLQLLLGNP; SEQ ID NO: 2) ofhuman RIP140, and measuring the proximity of the two labels, whereinbinding of the labeled peptide identifies the compound as an antagonistand displacement of the labeled peptide identifies the compound as anagonist.

In FIG. 4B, the addition of OR-942 causes the recruitment of peptide K(ERRTVLQLLLGNPTK; SEQ ID NO: 4), a 15-mer identical to amino acidresidues 708-722 of transcriptional coregulatory protein human RIP140(Iannone, Consler et al. 2001), to the RORγ LBD and increases the FRETsignal. In the presence of OR-942, T0901317 depresses the FRET signal.Further, T0901317 causes a rightward shift in the dose-response curvefor OR-942 (FIG. 5) as would be expected if both compounds compete forthe same binding site.

A modified coregulatory peptide was identified for the biochemicalassay, peptide K1 (ERRTVLQLLLGNSNK; SEQ ID NO: 3). In addition to itsmodified sequence, the peptide K1 reagent is attached to biotin by a sixcarbon linker that may facilitate better accessibility of biotin withstreptavidin in the peptide/receptor complex. K1 interacts withGST-mRORγ in the absence of agonist, and therefore we also used thebiochemical assay in this format in some of these experiments. In K1,the N-terminal biotin is linked to the first amino acid via6-aminohexanoic acid (AHC), which is introduced by usingFmoc-6-aminohexanoic acid (CAS NO: 88574-06-5) during peptide synthesis.In peptide K, the N-terminal biotin is directly linked to the firstamino acid without any linker.

As shown in FIG. 6, this assay could be used to characterize both anagonist (OR-12872) and an antagonist (OR-1050) independently under thesame assay conditions. The methods illustrated in FIG. 5 and FIG. 6 mayalso be modified to allow high throughput screening of RORγ agonists andantagonists with the coregulatory peptide recruitment assay.

A number of analogs of T0901317 and OR-1050 were synthesized andcompared in the transcriptional and biochemical assays for RORγ (Table1). These findings showed that the rank order of potency of the seriesof related molecules was similar in the two assays. Further, severalanalogs of OR-942 were also synthesized, including OR-12872, an agonistwith potency less than 100 nM in the biochemical assay (Table 2). Therank order of potency of these compounds was also similar in thetranscriptional and biochemical assays. In one embodiment, the RORγantagonist is not T0901317.

TABLE 1 (Structure 1)

Non-steroidal analogues of T0901317 Assay EC₅₀ in micromolar at (seeStructure 1) mRORγ X R1 R2 Transcriptional Biochemical OR-1048 OH Br Me0.12 0.42 OR-1052 OH Me Me 0.21 0.89 OR-1050 OH NO2 Me 0.26 0.9 T0901317OH H CF3 0.54 1.2 OR-1031 OH H Me 1.4 3.5 OR-1047 OH nBu Me 3.6 5.8OR-1030 OMe H H >20 >100 OR-1046 OH H CO2H >20 >100 The biochemicalassay was carried out in the presence of mRORγ and peptide K1.

TABLE 2 (Structure 2)

Analogues of OR-942 (see Structure 2) EC₅₀ in micromolar at mRORγ XTranscriptional Biochemical OR-12872 CH(Me)OMe 0.44 0.052 OR-12866C(Me)2OMe 0.56 0.14 OR-942 CO2Me 1.5 0.12 OR-12863 CH2OH 4.1 0.67OR-12870 C(═O)Me 4.1 ND OR-12868 C(═O)NMe2 5.2 0.56 OR-12864 CH2OMe 60.23 OR-12865 C(Me)2OH 8.3 0.21 OR-12871 CH(Me)OH 8.3 0.25 OR-412CO2H >20 >100 OR-12867 C(═O)NHMe >20 ND OR-12869 C(═O)N(c-C5H10) >20 NDDerivatives of hyodeoxycholic acid methyl ester (OR-942) were evaluatedin the transcriptional assay for RORγ and in the biochemical assay usingbiotinylated peptide K as the coregulatory peptide. In thetranscriptional assay, antagonist OR-376 (1.5 μM) was added to reducethe basal transcriptional activity or RORγ thus to increase the dynamicrange of the agonist effect. EC₅₀ values were estimated by fitting thedata to a sigmoidal curve (GraphPad, Prism, San Diego, CA). In thebiochemical assay, the compounds induce association between the RORγ LBDand peptide K. The EC₅₀ of the biochemical assay was determined by theconcentration of compound required to elevate the baseline by 50% ofestimated maximum FRET signal for the individual compound after fittingdata to a sigmoidal dose response curve.

In addition, several other RORγ antagonists were identified, includingthe related terpenes OR-345 and OR-885 (rockogenin) in Table 3. Thenatural compound OR-13571 (11-oxo ursolic acid acetate), is illustratedin Tables 4A and 4B. Further, the structurally-distinct syntheticcompound OR-2161 was also examined in RORγ assays.

TABLE 3 (Structure 3)

  In which X = O or is absent (X = H,H) Steroidal Antagonists Assay EC₅₀in micromolar X transcriptional biochemical OR-885 H,H 0.2 2.5 OR-345 O5 12.7 The biochemical assay was carried out in the presence of peptideK and 1 μM OR-942 to elevate the assay baseline. The EC₅₀ of thebiochemical assay was determined by the concentration of compoundrequired to inhibit the OR-942 baseline by 50% after fitting data to asigmoidal dose response curve.

TABLE 4A RORγ antagonists. EC₅₀ EC₅₀ Compound ID StructureTranscriptional Biochemical OR-13571

0.27 1.2 OR-2161

0.33 4.5 OR-133008

1.46 1.61 OR-133097

1.44 1.34 OR-133099

1.04 1.33 OR-133167

3.3 3.4 OR-133171

0.49 0.44 Biochemical assays were carried out in the presence of peptideK1. No agonist was present. OR-13571 is 11-oxo ursolic acid acetate.Values are medians of two or more assays.

TABLE 4B OR-52 and analogues of OR-52. Structure 4

Compounds (see Structure 4) Assay EC₅₀ in micromolar X R1 R2 R3Transcriptional Biochemical OR-52 CH2CH2 CF3 H H 5.8 3.5 OR-32286 CH2CF3 H H >20 >100 OR-32268 CH2CH2 CH3 H H 13.5 8.9 OR-32281 CH2CH2 Cl ClH 3.7 5.8 OR-32288 CH2CH2 CH3 H CH3 >20 52.2 Biochemical assays werecarried out in the presence of peptide K1. No agonist was present.

Further, many of the compounds listed above were evaluated in atranscriptional assay for hRORγ. As shown in Table 4C, the EC₅₀ valueswere comparable for most compounds tested, except for OR-885, which wassignificantly less potent at hRORγ, and the analogs of OR-133008, whichalso were more potent at mRORγ than at hRORγ.

TABLE 4C Potency of RORγ antagonists in a transcriptional assay of humanRORγ. Compound EC₅₀ (μM) OR-885 5.1 T0901317 1.1 OR-1050 0.4 OR-2161 1.0OR-13571 0.2 OR-133008 6.8 OR-133097 5.0 OR-133099 2.4 OR-133167 9.4OR-133171 1.7

These findings demonstrate that a range of ligands to RORγ may beidentified by receptor screening. The example also illustrates that aseries of agonists and a series of antagonists, each covering a widerange of potencies, will have consistent rank order of potencies in twomechanistically separate assays for the same target (e.g. mRORγ).Collectively, these findings strongly imply binding of these ligands tothe RORγ LBD. The most potent compounds, both agonists and antagonists,are also useful for characterization of RORγ-mediated effects in targetcells

Example 2

It is not unusual for a single compound to interact with receptors fromdifferent subfamilies within the nuclear receptor superfamily (Laudet1999). Nuclear receptor crossreactivity is sufficiently common that itmust be accounted for in a program of ligand design andcharacterization. An example of such a crossreactive ligand is TTNPB,which binds the retinoic acid receptors (RARα, RARβ, and RARγ) from theNR1B subfamily and the farnesoid X-receptor FXR (Zavacki, Lehmann et al.1997) in subfamily NR1H. Other examples are the estrogen receptor (NR3A)ligands diethylstilbestrol (DES), tamoxifen (TAM), and4-hydroxytamoxifen (4-OHT) which bind the estrogen-related receptor ERRγ(NR3B3) (Coward, Lee et al. 2001). Therefore, transcriptional assayswere developed to characterize RORγ ligands at several of the othermajor nuclear receptors.

Methods

Table 5 lists details of construction of Gal4 hybrids with the followingnuclear receptor LBDs. The positive control compounds fortranscriptional assays (Table 6) are dexamethasone, dihydrotestosterone,1,25 (OH)₂ Vitamin D₃, progesterone, chenodeoxycholic acid (CDCA), TTNPB((E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoicacid), 9-cis retinoic acid, 17β-estradiol, and bezafibrate (from Sigma,St. Louis, Mo.) and T0901317, rosiglitazone and WY14643 from CaymanChemicals. The transcriptional assays were carried out as in Example 1.

TABLE 5 The following nuclear receptor LBDs were cloned in pFA-CMV forexpression as Gal4 hybrids. Receptors are described by common namesfound in the literature, by a Unified Receptor Nomenclature system(Laudet 1999), and by species of origin (h = human or m = mouse) and atrivial name or abbreviation. The range of amino acids included in theLBD is shown along with the cloning site in pFA- CMV. The amino acidrange corresponds, in most cases, to the most common splicing isoform ofthe receptor. The final amino acid corresponds in each case to theC-terminal residue. Receptors were subcloned into pFA-CMV by PCR usingprimers containing the restriction enzyme sites shown. The methods arewell known to those skilled in the art and constructs similar to thesehave been widely reported in the scientific literature. Common UnifiedSpecies & Amino Name of Receptor trivial Acid cloning sites in ReceptorNomenclature name range pFACMV glucocorticoid NR3C1 mGR 524-793 BamHI,KpnI receptor androgen NR3C4 hAR 646-919 BamHI, XbaI receptor Vitamin DNR1I1 hVDR  90-427 BamHI, HindIII receptor Liver X NR1H3 mLXRα 172-455BamHI, XbaI receptor alpha progesterone NR3C3 hPR 687-933 XbaI, KpnIreceptor PPAR-gamma NR1C3 hPPARγ 175-477 XbaI, KpnI farnesoid X NR1H4hFXR 192-473 BamHI, XbaI receptor retinoic acid NR1B1 hRARα 156-462BamHI, HindIII receptor alpha retinoid X NR2B1 hRXRα 203-462 EcoRI,HindIII receptor alpha PPAR-alpha NR1C1 hPPARα 166-468 BamHI, HindIIIPPAR-delta NR1C2 hPPARδ 138-441 BamHI, HindIII Estrogen NR3A1 hERα249-595 BamHI, KpnI receptor-alpha

TABLE 6 Crossreactive receptor assays for RORγ ligands. Standard RORγLigands Standard Ligands OR- OR- OR- OR- Ligand Efficacy PotencyT0901317 OR-1050 OR-885 13571 12872 2161 133008 mGR Dexamethasone 35X++++ − − − − − − − hAR dihydro- 8X ++++ − − − − − − − testosterone hVDR1,25 Vitamin 68X ++++ − − − − − − − D3 mLXRα T0901317 120X ++ ++ + − − −− − hPR Progesterone 6X ++++ − − − − − − − hPPARγ Rosiglitazone 50X +++− − − − − − − hFXR CDCA 5X ++ + − − − − − − hRARα TTNPB 18X ++++ − − − −− − − hERα 17β-estradiol 40X ++++ − − − − − − − hRXRα 9-cis-retinoic180X +++ − − − − − − − acid hPPARα WY14643 6X * − − − − − − − hPPARδBezafibrate 10X * − − − − − − − Potency indicates EC50 values in μM:++++ (EC50 < 0.01), +++ (0.01-0.1), ++ (0.1-1), + (1-10) and − (>10). *EC₅₀ values of standard ligands for PPARα and PPARδ are >>20 μM.Efficacy = activity of standard ligand at 20 μM. The RORγ ligands showno significant activity at 20 μM in assays for PPARα and PPARδ.

Results

Among the RORγ ligands identified in studies described in Example 1, oneof the compounds, T0901317, was previously shown to activate LXRα andLXRβ (Schultz, Tu et al. 2000). A transcriptional assay for LXRα, usinga Gal4-LXRα hybrid, was implemented in order to compare RORγ ligandpotencies. In addition to LXRα, transcriptional assays for a number ofother major nuclear receptors were developed. In Table 6, several of theRORγ ligands described in Example 1 were characterized against thesereceptors. Confirming published data on T0901317 (Schultz, Tu et al.2000; Houck, Borchert et al. 2004), we found that T0901317 activates LXRand FXR (Table 6). At the same time, a number of other RORγ antagonists,such as OR-12872, OR-885, OR-13571, OR-133008, and OR-2161 were shown tobe selective for RORγ within this receptor family.

TABLE 7 Comparative pharmacology of RORγ antagonists at RORγ and LXRα.Compound structures are presented in Table 1, and RORγ pharmacology istaken from Table 1. Activity in the LXRα transcriptional assay is theestimated maximum elevation of luciferase activity, based on a sigmoidaldose response curve, with respect to T0901317. Assay EC₅₀ inTranscriptional RORγ micromolar at mRORγ Assay of LXRα antagonistsTranscriptional Biochemical activity EC₅₀ OR-1048 0.12 0.42 >80% 0.57OR-1052 0.21 0.89 >80% 0.81 OR-1050 0.26 0.9 >80% 1.5 T0901317 0.54 1.2100% 0.14 OR-1031 1.4 3.5 >80% 1.4 OR-1047 3.6 5.8  <5% >20OR-1030 >20 >100  <5% >100 OR-1046 >20 >100  <5% >100

In Table 7, several analogs of T0901317 were compared in transcriptionalassays for RORγ and LXRα. The binding pockets of LXRα and LXRβ are verysimilar in structure and have closely similar affinities to a range ofligands (Svensson, Ostberg et al. 2003). Hence, the LXRα assay isassumed to be representative of both receptors. A number of derivativesof T0901317 were synthesized. As can be seen in Table 7, compounds witha wide range of specificity were identified, including several that aremore specific for RORγ than LXRα, including OR-1048, OR-1050, OR-1052and OR-1047.

The importance of this example is two-fold. First, selective RORγligands can be identified that lack obvious cross-reactivity with othernuclear receptors. Secondly, the SAR of compounds in the T0901317 seriesdiffer between RORγ and LXRα, suggesting that ligands can be designedfrom this series that retain RORγ potency and have better separationbetween the two targets.

In one embodiment, the RORγ antagonist is at least 20-fold more potentas an RORγ antagonist than as an LXR agonist. In other embodiments, theRORγ antagonist is at least about 30-fold, 40-fold, 50-fold, 75-fold,100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 400-fold, 500-fold,600-fold, 700-fold, 800-fold, 900-fold, or 1,000-fold more potent as anRORγ antagonist than as an LXR agonist.

Ligands to LXR have anti-inflammatory effects that may make them activein animal models of autoimmune disease (Zelcer and Tontonoz 2006).Recently, T0901317 was shown to inhibit the development of murine EAE(Hindinger, Hinton et al. 2006). It is not known what proportion of thetherapeutic effect of T0901317 in the mouse model of EAE is due to RORγantagonism as opposed to LXR activation. However, strong genetic andpharmacological data demonstrate that activation of LXR causeshypertriglyceridema and triglyceride accumulation in liver.Specifically, in mice lacking the LXRα and LXRβ receptors, T0901317 hasno effect on liver triglyceridemia (Schultz, Tu et al. 2000). Other LXRligands have been tested in cynomolgus monkey and hamster (Groot, Pearceet al. 2005), and these ligands were shown to elevate serumtriglycerides and serum LDL. The importance of this example is that itprovides methodology to eliminate various forms of LXR-mediatedhyperlipidemic activity from the T0901317 and OR-1050 series ofcompounds while retaining the ability to inhibit T_(H)-17differentiation.

Example 3

The differentiation of purified naïve murine CD4⁺ splenic T cells intoT_(H)-17 cells in cell culture is simulated by a combination of thecytokines TGFβ and IL-6 or inhibited by the cytokine IFN-γ (Mangan,Harrington et al. 2006; Veldhoen, Hocking et al. 2006). This is not theonly differentiation pathway available to naïve CD4⁺ T cells (FIG. 1).For example, IL-12 stimulates the differentiation of T_(H)1 cells in thesame system. The effects of continuous treatment with RORγ agonists andantagonists on T_(H)1 and T_(H)-17 differentiation were investigated.This example demonstrates that small molecule ligands to RORγ regulateT_(H)-17 T cell differentiation. In these experiments, a T_(H)-17 cellwas identified by cell surface staining for CD4 and intracellularstaining with a labeled antibody to IL-17. In particular, we observethat antagonists to RORγ inhibit T_(H)-17 cell differentiation but haveno consistent effect on the differentiation of murine T_(H)1 cells (thatare CD4⁺ IFNγ⁺ but do not express IL-17). The inhibitory effect onT_(H)-17 cells has been observed with four different antagonists thatare each structurally distinct (that is, they are not analogs of oneanother). Further, supporting the conclusion that inhibition of RORγactivity in naïve CD4⁺ T cells by an RORγ antagonist will inhibitT_(H)-17 differentiation, we show that an agonist to RORγ reversesantagonist inhibition of T_(H)-17 cell differentiation and independentlyenhances T_(H)-17 cell formation. These results indicate that the RORγligands described in the example act specifically through the RORγ LBDin the differentiating T_(H)-17 cell.

Methods

Antibodies and Cytokines.

The following reagents were selected for characterization of mouselymphocytes. FITC anti-IFNγ (XMG1.2), PE anti-CD62L (MEL-14), APCanti-CD4 (RM4-5), FITC anti-CD4 (RM4-5), PE anti-CD8 (53-6.7), APCanti-CD25 (PC61), biotinylated anti-CD25 (PC61.5), Cy5.5 anti-CD44PE-(IM7) were from eBioscience (San Diego, Calif.). PE anti-IL-17(TC11-18H10.1) was from BD Biosciences (San Diego, Calif.). TGF-β andIL-6 were from Sigma. IL-12 was from PeproTech (Rocky Hill, N.J.).

Purification of Naïve (CD4⁺CD62L+CD25⁻) CD4 Cells. Spleens of 5-7 weekold outbred ICR (CD-1, Harlan Labs) mice were gently minced with two18-gauge needles in PBS buffer supplemented with 2.5 mM MgCl₂, 0.5 mMCaCl₂ and 25 ug/ml DNAse I. A single cell suspension of splenocytes wastriturated with a 5-ml pipet and passed through a 40 μm nylon meshfilter. Splenocytes were spun down at 300×g for five minutes andresuspended in buffer containing PBS, 2% heat-inactivated FCS and 2 mMEDTA. Naïve T cells were purified from splenocytes using a CD4⁺CD62L⁺magnetic bead T cell Isolation Kit from Miltenyi Biotec (Auburn, Calif.)in two steps. First, CD4⁻ cells were removed by negative selection withbiotinylated antibodies to CD8a, CD45R, CD11b, CD49b, and Ter119(supplied by the manufacturer) and to CD25 (biotinylated PC61), added atthe time of isolation. The flow through, predominantly CD4⁺ cells, wasdirectly labeled with anti-CD62L microbeads and enriched on a magneticcolumn to about 93% purity, as determined by staining for CD4, CD62L,and CD25.

Stimulation and Culture of T_(H)-17 Cells.

Purified CD4⁺CD62L⁺ T cells were plated in 24-well plates with 2-4×10⁵cells in 700 ul volume of RPMI 1640 media containing 10%heat-inactivated FCS, 100 IU penicillin, 100 μg/ml streptomycin, 1×non-essential amino acids, 1 mM pyruvate, 2 mM glutamine, and 50 μMβ-mercaptoethanol (Veldhoen, Hocking et al. 2006). T cells werestimulated with exogenous cytokines and anti-CD3/anti-CD28 conjugatedDynabeads (Dynal Biotech, Oslo) at a 1:1 ratio of beads to cells.Exogenous cytokines used were: 4 ng/ml TGF-β, 20 ng/ml of IL-6, or 10ng/ml IL-12. RORγ agonist and antagonist ligands, dissolved in DMSO,were added directly to cell cultures, to a final concentration of 0.01%.Cytokines and RORγ ligands were added immediately after cell plating.

Cell Staining and Flow Cytometry.

To determine intracellular cytokine production, exogenous cytokines wereremoved by centrifugation and cultures treated with 500 ng/ml phorbol12,13 dibutyrate (PdBu), 500 ng/ml ionomycin, and 1 μg/ml brefeldin Afor 4-6 hours to stimulate cytokine production while preventingsecretion as described (Veldhoen, Hocking et al. 2006) and washed again.Cells were then fixed, permeabilized and stained for intracellularcytokines using commercial reagents (eBioscience, San Diego). Cells werealso stained for the cell surface markers CD4 and CD8. Flow cytometryanalysis was performed on a FACSCalibur (Becton Dickinson) and the datawere analyzed using FlowJo Software (Tree Star, Inc).

ELISA for IL-17.

ELISA kits for mouse IL-17 and human IL-17 were from eBioscience (SanDiego, Calif.). ELISA assays were performed following the manufacturer'sprotocol. Certain samples were diluted 20-fold to avoid maximization ofenzymatic signals. For mouse IL-22 ELISA, antibodies and standards werefrom Antigenix America (Huntington Station, N.Y.) and accessorycomponents were from eBioscience. The IL-22 assay protocol was similarto that of the IL-17 assays.

Results

Mouse T_(H)-17 cells can be induced to differentiate from naïve CD4⁺ Tcells in the presence of TGFβ and IL-6 (Veldhoen, Hocking et al. 2006).At the same time, T_(H)1 differentiation is suppressed. Critical stepsin this protocol are: (i) purification of naïve CD4⁺ cells to removepotentially inhibitory activated T cells, such as T_(H)1 cells, or Tregulatory (Treg) cells, (ii) activation of T cells by crosslinking CD3and CD28, and (iii) incubation with TGFβ and IL-6 to induce T_(H)-17differentiation. Protocols for T_(H)-17 differentiation have beenreported by several laboratories (Bettelli, Carrier et al. 2006; Mangan,Harrington et al. 2006; Veldhoen, Hocking et al. 2006). The percentageof differentiated cells is determined by intracellular staining forIL-17 and IFN-γ, markers of T_(H)-17 and T_(H)1 cells respectively(Park, Li et al. 2005). A T_(H)-17 differentiation assay provides acell-based model to demonstrate RORγ antagonist function. We predictedthat OR-1050, OR-885, OR-13571, and OR-2161 should inhibit T_(H)-17differentiation and/or IL-17 release and that this inhibitory effectcould be reversed by the potent RORγ agonist OR-12872.

Naïve CD4⁺ T cells were isolated from splenocytes of 5-8 wk old CD-1mice and CD4⁺CD62L⁺ cells with approximately 93% purity selected withmagnetic beads. We estimated that the population of Tregs (Itoh,Takahashi et al. 1999) in the purified cells, as estimated by thepercentage of CD25+CD44⁺ cells, was about 1-1.5% as compared to 5% intotal splenocytes. IL-12 treatment of the naïve T cells stimulatesT_(H)1 differentiation as shown by the high percentage of IFNγ positivecells, while the combination of TGFβ1 and IL-6 induced the formation of4% IL-17-producing cells (Table 8). As reported, the T_(H)1 (IFNγ⁺) andT_(H)-17 (IL-17⁺) phenotypes were mutually exclusive (see also FIG. 8).Further, both IL-6 and TGFβ were required to fully induce the T_(H)-17phenotype as reported in the scientific literature (Bettelli, Carrier etal. 2006; Mangan, Harrington et al. 2006; Veldhoen, Hocking et al.2006). Finally, we observed that the IL-17⁺ cells were >97% CD4⁺.

TABLE 8 IL-17- or IFNγ-producing cells are differentiated from naïve CD4cells in the presence of exogenous cytokines. Cells are stained forIL-17 or IFN-γ and quantitated by FACS analysis. Data are shown as mean± SD of the percentage of IL-17 or IFNγ-producing cells in total cellpopulations after 5 days in culture. Less than 0.02% of total cellsappear to express both cytokines. IL-17 (%) IFNγ (%) No cytokine 0.21 ±0.00 2.50 ± 0.40 IL-12 (10 ng/ml) 0.09 ± 0.01 39.6 ± 2.44 TGFβ1 (4ng/ml) 0.21 ± 0.02 0.32 ± 0.05 IL-6 (20 ng/ml) 1.20 ± 0.04 3.42 ± 0.08TGFβ1 + IL-6 4.53 ± 0.18 2.14 ± 0.18

For pharmacology studies, we added either agonist or antagonist to RORγat the initiation of naïve CD4⁺ T cell cultures described above. Wefound that OR-1050 significantly inhibited T_(H)-17 cell differentiationin a dose-dependent manner while the potent agonist OR-12872 wasstimulatory (see FIG. 7).

As expected from receptor pharmacology studies, OR-12872 was able toreverse the effect of OR-1050, consistent with the conclusion that bothcompounds act through the same target, i.e., RORγ (FIG. 8). In thisexperiment, the agonist OR-12872 alone elevated T_(H)-17 frequency by60%, but in the presence of OR-1050 the agonist caused a 400% percentincrease from the lower baseline produced by OR-1050, precisely thebehavior expected if agonist and antagonist bind the same target. Thefigure represents individual observations, and the average of duplicateobservations is presented in Table 9. Neither 3 M OR-1050 nor 3 μMOR-12872 had an effect on the frequency of T_(H)1 cell differentiationin the presence of IL-12. The findings show that OR-1050 is unlikely toblock T_(H)-17 cell formation through a non-specific cytotoxic effectfor two reasons: it does not inhibit T_(H)1 differentiation and theblockage of T_(H)-17 differentiation is reversible by an RORγ agonist.

TABLE 9 Naïve CD4+ T cells were induced to differentiate with 4 ng/mlTGFβ and 20 ng/ml IL-6 (T_(H)-17 protocol) or 10 ng/ml IL-12 (Th-1protocol). After addition of compound, the final DMSO concentration inthe culture wells was 0.01%. Cytokine positive cells were determined asin the methods for this example. Duplicate cell cultures were countedand averaged. Cell Frequency (%) T_(H)-17 Protocol Th-1 ProtocolCompound IL-17⁺ IFN-γ⁺ IFN-γ⁺ DMSO 2.32% 1.60% 36.3% 3 μM OR-1050 0.44%1.42% 37.4% 3 μM OR-12872 3.91% 1.37% 41.4% OR-1050 + OR-12872 1.79%1.47% 36.6%

To provide additional confirmation that T_(H)-17 cell differentiationcan be pharmacologically-regulated through inhibition of RORγ function,we tested the effects of the antagonist OR-885 and its reversal byOR-12872. FIG. 9 demonstrates that OR-885 blocks the differentiation ofT_(H)-17 cells and that this effect is reversed by OR-12872. The resultsof this experiment are summarized in Table 10. It appears that there maybe a small secondary effect of ligand on Th-1 cell levels. Overall,however, the RORγ ligands did not have a consistent effect on Th-1 cellfrequency in these studies.

TABLE 10 Naïve CD4+ T cells were induced to differentiate with 4 ng/mlTGFβ and 20 ng/ml IL-6 (T_(H)-17 protocol) or 10 ng/ml IL-12 (Th-1protocol). After addition of compound, the final DMSO concentration inthe culture wells was 0.01%. Cytokine positive cells were determined asin the methods for this example. Duplicate cell cultures were counted.Cell Frequency (%) T_(H)-17 Protocol Th-1 Protocol Compound IL-17⁺IFN-γ⁺ IFN-γ⁺ DMSO vehicle 2.46% 0.71% 29.7% 3 μM OR-885 0.52% 1.45%34.2% 1 μM OR-12872 3.96% 0.65% 30.8% OR-885 + OR-12872 1.33% 1.11%36.2%

Finally, OR-13571 exerted a similar effect on T_(H)-17 differentiationas OR-885 and OR-1050. The effect of 3 μM OR-1050 was compared to thatof 3 μM OR-13571 in Table 11.

TABLE 11 Studies were performed as in Table 9. Cell Frequency (%) Th-17Differentiation Compound IL-17⁺ IFN-γ⁺ DMSO vehicle 3.81% 1.03% 3 μMOR-1050 1.47% 1.09% 3 μM OR-13571 1.42% 0.93%The data show that OR-13571 also inhibits T_(H)-17 differentiation at aconcentration of 3 M. In summary, this example provides very strongpharmacological evidence that regulation of RORγ activity by smallmolecule ligands can control cell fate. Antagonists with 3 distinctstructures were shown to have a similar effect on T_(H)-17differentiation and two of these were reversed by a specific RORγagonist, OR-12872. It is probable that the RORγ ligands will regulateT_(H)-17 function in other settings described in the literature,including by measurement of IL-17 release into surrounding medium and byculture of cells from lymph nodes of animals treated with antigen thatprovokes autoimmune disease (Murphy, Langrish et al. 2003; Langrish,Chen et al. 2005; Mangan, Harrington et al. 2006). This hypothesis isaddressed in Example 6.

We also investigated whether the concomitant release of IL-17 that isexpected to take place with T_(H)-17 differentiation ispharmacologically regulated by RORγ ligands. RORγ antagonists at 3 μMwere added as naïve CD4⁺ T cells were induced to differentiate intoT_(H)-17 cells, in the presence or absence of OR-12872. In this study,all four RORγ antagonists tested block IL-17 release (FIG. 10).Significantly, the presence of OR-12872, the RORγ agonist, reverses theeffect of each of the antagonists, indicating that the compounds areacting in an RORγ-specific manner. We hypothesize that the very markedreduction in IL-17 levels involved simultaneous inhibition of T_(H)-17differentiation, as shown above, and suppression of IL-17 release.

Example 4

This example provides data suggesting that pharmacological repression ofRORγ will not necessarily inhibit thymic function in vivo. The inductionof liver triglycerides by OR-1050 in C57BL/6 mice following oral dosingwas investigated. In addition, the effect of compound treatment in thesame animals on the number and distribution of the major thymocytepopulations was analyzed. The objective was to show evidence ofbioavailability through elevation of liver triglycerides (through LXR),and at the same time to characterize the effect of OR-1050-mediatedantagonism of RORγ on thymic function.

Methods

Following six days of twice-a-day dosing with corn oil vehicle or 50mg/kg OR-1050 in corn oil, mice were sacrificed and the thymus and liverremoved and weighed. A small piece of the liver (˜200 mg) was weighedand extracted to determine triglyceride content. Glycerol was liberatedfrom liver triglycerides by hydrolysis in base. The liver fragment wasincubated overnight at 55° C. in 0.35 mls of ethanolic KOH (2 partsEtOH: 1 part 30% KOH) in a closed tube. A solution of 50% ethanol inwater was added to bring the volume of each tube to 1.0 ml. The solutionwas cleared of debris by centrifugation, and the resulting supernatantincreased to 1.2 ml with further addition of 50% ethanol. Aqueous 1 MMgCl₂ (215 μl) was added to 200 μl of the supernatant, and the mixturewas cleared by centrifugation again after standing 10 min on ice.Glycerol levels were measured with the Sigma Free Glycerol Reagent(Sigma-Aldrich, St. Louis, Mo.) according to the manufacturer'sinstructions. The triglyceride concentration (in milligrams per gram ofliver wet weight) was determined by assuming average molecular weight of1000 daltons for each molecule of triglyceride.

In addition, the total number and the fraction of the major thymocytesubpopulations including CD4⁺CD8⁺ (DP), CD4⁺CD8⁻ (SP4⁺) and CD4⁻CD8⁺(SP8⁺) were measured by cell surface staining followed by FACS analysis.The thymus was dissected, weighed, and gently pressed against a wirescreen (200 mesh USA standard test sieve, Newark Wire Cloth Company)with plunger of a 5 ml plastic syringe. The volume of cells was adjustedto 8 mls. Debris from the thymus was allowed to settle, and a 200 μlaliquot of cells was incubated with fluorescent antibodies to CD4 andCD8 for 15 minutes at room temperature. The cells were fixed in 4%formalin in PBS and stored at 4° C. for 48 hours. Before analytical flowcytometry was carried out (FACS Calibur, Becton Dickinson), Caltagcounting beads (from Invitrogen) were added to allow absolutequantitation of cell number. Thymus subpopulations were determined aftergating for live cells. As a positive control for thymic involution, twoanimals were also treated once with 10 mg/kg dexamethasone two daysbefore sacrifice. Dexamethasone reproducibly induces rapid apoptosis ofDP thymocytes and reduction of thymic mass (Chmielewski, Drupt et al.2000; Zubkova, Mostowski et al. 2005).

Results

Table 12 shows that liver triglyceride content (in mg oftriglyceride/gram liver) is markedly elevated by OR-1050 as reported forother LXR agonists (Schultz, Tu et al. 2000; Beyer, Schmidt et al.2004). In contrast, the number and fraction of major thymocytesubpopulations was not significantly affected. As expected,dexamethasone caused a virtually total depletion of DP thymocytes.

As shown above, OR-1050 is approximately 5-fold more potent inrepression of RORγ transcription than it is in induction of LXRtranscription. Thus it is likely that RORγ activity in mice treated asabove was significantly repressed. This example demonstrates thatpharmacological dosing of an RORγ antagonist does not necessarily causethymic atrophy or loss of thymocytes following an intermediate period ofdosing.

TABLE 12 Effects of OR-1050 on liver triglycerides and thymocytedistribution. C57BL/6 mice were treated by vehicle only (corn oil) orOR-1050 (100 mg/kg) for 7 days, or by dexamethasone (Dex, 12.5 mg/kg)once 2 days before necropsy. Corn oil and OR-1050 (50 mg/kg) were dosedtwice per day by gavage. Liver triglycerides are reported as mgtriglyceride/gram liver wet weight. One animal from the OR-1050 grouphad a very low frequency of DP thymocytes (<1% of control) and wasexcluded from this analysis. Frequency Liver Thymus Total cells perspleen of DP (% Group triglycerides weight (×10⁻⁶) of total size (mg/g)(μg) DP SP4 SP8 cells) Vehicle 6 34.7 ± 5.7  71.3 ± 12.0 58.7 ± 24.7 6.3± 1.7 2.0 ± 0.4 52.6 ± 8.4 Dex 2 35.3 ± 8.2 49.7 ± 1.2 1.0 ± 0.4 3.9 ±0.1 1.2 ± 0.1  4.2 ± 2.9 OR-1050 5 119.5 ± 18.8 76.1 ± 5.8 61.5 ± 10.08.1 ± 1.6 2.5 ± 0.5 56.7 ± 3.1

Example 5

This example provides a demonstration that an RORγ antagonist mayinhibit the development of EAE in a mouse model.

Methods

8-10 week old, female C57BL/6 mice or SJL/J mice were purchased fromHarlan laboratories (Harlan, Indianapolis, Ind.) and housed in aspecific pathogen free (SPF) animal facility for one week before thestart of the studies. Mice were immunized with peptide antigensintradermally at the dorsal flanks on day 0. Peptide antigens are either150 g MOG₃₅₋₅₅ (MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 19), AnaSpec, SanJose, Calif.) per C57BL/6 mouse or 75 μg PLP₃₉₋₁₅₁ (HSLGKWLGHPDKF (SEQID NO: 20), Bio-Synthesis, Lewisville, Tex.) per SJL/J mouse. Peptideswere dissolved in PBS and emulsified with an equal volume of CompleteFreund's Adjuvant (CFA) containing 4 mg/ml of H37RA M. tuberculosis(Chondrex, Redmond, Wash.). To ensure induction of reliable EAE, 200 ngof pertussis toxin (List Biologicals, Campbell, Calif.) was given by viaintravenous injection (i.v.) at day 0 and 2 (Cua, Sherlock et al. 2003;Zhang, Gran et al. 2003). Animals were treated with drug beginning oneday before peptide injection. Animals were randomized into groups fortreatment by weight, so that the average weight of each group wassimilar. The groups were not segregated by cage. Instead, members of allgroups were represented in every cage in order to minimize environmentaleffects on the outcome of the study. In the C57BL/6 study, OR-1050 wassuspended in corn oil by sonication at 6 mg/mL or 20 mg/mL and animalswere dosed twice daily by gavage with 2.5 mls/kg body weight of corn oilfor each dose. In one SJL/J study (FIGS. 11 D & E), OR-1050 wasdissolved in dimethylformamide at 200 mg/ml, further diluted into threeparts of HRC-6, loaded into an osmotic pump (Alzet 2002) and implantedin the interscapular region under anesthesia 2-3 days after peptideimmunization. The estimated daily does was 30 mg/kg in this study. Inanother SJL/J study (see FIG. 11F), mice were treated once daily bygavage with 100 mg/kg OR-1050 solubilized in the synthetic vehicleHRC-6.

Clinical symptoms are scored by a visual inspection of behavior, alongthe following scale (Papenfuss, Rogers et al. 2004): 1) Limp tail orwaddling gait; 2) waddling gait with tail limpness; 3) partial hind limbparalysis; 4) complete hind limb paralysis; 5) moribund state, leadingto death by EAE. At severity level 4 or 5 the animals were sacrificed toprevent further suffering. Animals were weighed daily during thetreatment period as a secondary measure of the disease course. As acomparative measure for drug treatment, the median day of onset wascompared between groups. Statistical analysis was by the Kruskal-Wallistest, a non-parametric test for comparison of multiple groups. On studyday 27, remaining animals were euthanized, and brain and spinal cordfixed, embedded in paraffin, and H&E sections analyzed for inflammatoryinfiltration. For the studies of inflammation, several sections ofspinal cord (7 to 8) were characterized by an experienced veterinarypathologist in a blinded fashion. Inflammation in each section wasevaluated visually using a semi-quantitative scale based on the overallseverity of the sample (e.g., Severity: 0, not present; 1, slight; 2,mild, 3, moderate; 4, severe).

Results

C57BL/6 mice were treated with OR-1050 by oral gavage twice a day for atotal dose of 30 mg/kg or 100 mg/kg for 25 days. Mice were injected witha peptide that induces autoimmune disease in this mouse strain one dayafter the start of dosing. The progress of the disease in the vehicleand high dose groups is illustrated in FIG. 11A and FIG. 11B, whereexposure to OR-1050 both inhibits progress of the disease and weightloss doe to the onset of symptoms. FIG. 11C shows that inflammatoryinfiltration in the spinal cord was reduced by treatment with OR-1050 inthese animals. The onset of disease in the three groups as a function ofnumber of days since peptide immunization is presented in FIG. 12. Themedian day of onset of symptoms (clinical score 1 or greater) was day 18for the corn oil vehicle control, day 16 for the 30 mg/kg group, and day26 for the 100 mg/kg treated group (FIG. 12A). The median day of onsetfor untreated animals was day 15 (not shown). The difference in day ofonset between the vehicle and the 100 mg/kg treated animals wasstatistically significant (p<0.05). This example shows that an RORγantagonist is able to delay the onset of EAE. The surviving animals atday 27 showed a difference in the severity of inflammation in spinalcord between vehicle and high dose treated animals. The statisticalsignificance was based on a rank order test (p<0.07, Mann-Whitney).

In a second EAE mouse study, SJL/J mice were immunized with PLP139-151and OR-1050 was dosed by osmotic pump to achieve more stable compounddelivery starting at day 4. OR-1050 also reduced paralytic severity(FIG. 11D) and onset (FIG. 11E). In each of these studies, there was asignificant difference in severity or weight on several days, asindicated in the graphs (FIGS. 8A, B&D). Using a more rigorousstatistical analysis, we calculated the area under the severity curvefor each of the mice in FIG. 11D, and compared the mean values±sem forvehicle (14.5±1.6) and treated (5.3±2.7) groups. The differences weresignificant (p<0.01) based on the Mann-Whitney rank order test (GraphPadPrism, San Diego). Furthermore, as shown in FIG. 11E, the day of onsetwas significantly delayed in the treated SJL/J mice.

Since OR-1050 inhibits T_(H)-17 cells in ex vivo culture, we alsoinvestigated whether T_(H)-17 cell frequency was affected inOR-1050-treated animals. SJL/J female mice (n=9/group) were treated with100 mg/kg OR-1050 in HRC-6 beginning two days before immunization withPLP (as described above) and continuing until 8 days after immunization.Animals were sacrificed on day 9, and T_(H)-17 cells analyzed frompooled axillary, brachial, and inguinal lymph nodes from each animal. Wefound a 35% reduction in T_(H)-17 frequency in lymph nodes of SJL/J mice(FIG. 11F) as a fraction of the total CD4⁺ cell population. The changein T_(H)-17 frequency with OR-1050 treatment was statisticallysignificant.

In a second study on C57BL/6 mice (FIG. 1G), we observed a similareffect on splenic T_(H)-17 cells in C57BL/6 mice (29% inhibition,p<0.05, n=8-10) with no change in T_(H)1 cells. C57BL/6 female mice wereimmunized with 150 μg MOG₃₃₋₅₅ on study day 0. Mice were treated witheither OR-1050 at 100 mg/kg or with vehicle (HRC-6) by oral gavage for 9days starting day 0. Splenocytes were collected at Day 9 and stained forT_(H)-17 (CD4⁺CD8⁻IL17⁺) and T_(H)1 (CD4⁺CD8⁻IFNγ⁺) cells. Statisticswere performed by Student's t-test. Collectively, the data suggest thatOR-1050 inhibits the expansion of T_(H)-17 cells in response to peptideantigen.

In a separate study related to these findings, we demonstrated thatOR-1050 is orally bioavailable (FIG. 12B). Pharmacokinetics of OR-1050in CD-1 mice was analyzed at independent bioanalytical laboratory. MaleCD-1 mice were treated via oral gavage with OR-1050 at 5 mg/kg in HRC-6.Blood was collected via retro-orbital puncture in tubes containingsodium heparin anticoagulant at 0, 5, 15, and 30 min, and at 1, 2, 4, 6,8 and 24 hr after oral administration. The concentration of OR-1050 inplasma was determined using an HPLC/MS/MS method. The peak concentrationof OR-1050 (˜100 ng/ml) suggests that a higher dosage (50 mg/kg) willgive blood levels, 1 μg/ml or ˜2 μM, that is higher than the EC₅₀ forOR-1050 in transcriptional assays in in vivo pharmacological studies.

Example 6

That RORγ antagonists inhibit the differentiation of T_(H)-17 isconsistent with the phenotype of the RORγ^(−/−) mouse, which lacksT_(H)-17 cells and where naive CD4⁺ T cells resist differentiate toT_(H)-17 in the presence of IL-6 and TGFβ (Ivanov, McKenzie et al.2006). The question of whether the reactivation of memory T_(H)-17 cellscan be blocked by RORγ ligands is not addressed by the RORγ^(−/−) mousephenotype, since memory T_(H)-17 cells are unable to differentiate fromnaïve CD4⁺ T cells. To address the issue, we cultured lymph node cellsfrom mice previously immunized with the myelin-derived peptidesPLP₃₉₋₁₅₁, MOG₃₅₋₅₅, or complete Freund's adjuvant (CFA) alone. Memory Tcells are known to proliferate in response to cognate peptide in thesecultures, and our controls showed that stimulation of T_(H)-17proliferation and IL-17 release were dependent on prior immunizationwith peptide, consistent with a memory T cell response (FIG. 13A). Thus,LN cultures from CFA-immunized mice were insensitive to PLP in 3 dayculture, whereas LN cultures from mice immunized with PLP (emulsified inCFA) showed a ˜10-fold increase in IL-17 release and a concomitanttwo-fold elevation in the frequency of T_(H)-17 cells in response topeptide (not shown). These data show that IL-17 induction is closelylinked to the peptide-dependent increase in T_(H)-17 cells and thereforeprovides a convenient method of characterizing compound effects on Th-17cells. Furthermore, OR-885 inhibited the PLP-stimulated release ofIL-17. The correlation between Th-17 cell number and IL-17 release inthe presence of agonist and antagonist was further confirmed (FIGS. 13Band 13C), and the antagonist OR-885 was shown to block both theinduction of T_(H)-17 cells (as a percentage of total live cells) andIL-17 release.

Overall, cultured lymph node cells that are stimulated with IL-23 andpeptide antigens provide a model system to examine IL-17 production exvivo (FIG. 13D). We analyzed a variety of RORγ ligands for their abilityto regulate IL-17 (FIG. 13E). Four different classes of RORγ antagoniststested all inhibit IL-17 release, while the agonist is able to activateIL-17. A dose-response assay suggested that the antagonist OR-885inhibits IL-17 production with an EC₅₀ value of approximately 0.2 μM(FIG. 13F). Each point is the average of duplicate measurements of IL-17release in a single well. This potency is consistent with that measuredin the CHO cell transcription assay. To further confirm that theactivity of compounds was RORγ-dependent in this system, we showed thatthe agonist OR-12872 reverses the suppressive effects of OR-885 on IL-17release in lymph node cultures (FIG. 14).

The proinflammatory cytokine IL-22 is also expressed in Th-17 cells. Wecharacterized IL-22 release from lymph node cell cultures and found thatlike IL-17, IL-22 release is induced by both peptide antigen and IL-23,and that the effect of both was more than additive. In addition, IL-22seems to have 30-50 fold higher levels than IL-17. IL-22 production isalso inhibited by the RORγ antagonists OR-885 and OR-1050 and enhancedby the agonist OR-12872 at a concentration of 3 μM each in LN cultures,similar to findings with the IL-17 assays. This is illustrated in FIG.15.

Regulation of Human IL-17 Release in Cultured PBMCs.

We also investigated whether human IL-17 is subject to regulation byRORγ ligands. It has proven difficult to differentiate Th-17 cells fromnaïve human T cells under the same conditions as mouse (Zheng, Danilenkoet al. 2007), although there are now several very recent reports thatthis can be done (Acosta-Rodriguez, Napolitani et al. 2007; Annunziato,Cosmi et al. 2007; Wilson, Boniface et al. 2007). Since mitogenicstimuli such as ConA or PMA will stimulate IL-17 expression in memory Tcells in human PBMC (Yao, Painter et al. 1995; Shin, Benbernou et al.1999), we used this protocol for an initial investigation of IL-17regulation by RORγ ligands. Frozen human peripheral blood mononuclearcells (PBMCs from Stem Cell Technologies, Vancouver) were thawed andcultured for 3 days with 1 μg/ml Con A and 3 μM compounds in lymphocyteculture medium (RPMI 1640 media containing 10% heat-inactivated FCS, 100IU penicillin, 100 μg/ml streptomycin, 1× non-essential amino acids, 1mM pyruvate, 2 mM glutamine, and 50 μM β-mercaptoethanol). Culture mediawere analyzed by hIL-17 ELISA (mean±sem, n=4; ANOVA with Bonferroni posttest). In FIG. 16A, the effects of 3 μM OR-885 and 3 μM OR-12872 onIL-17 release from PBMCs were compared. The effect of OR-885 waspartially reversible by OR-12872. FIG. 16B shows that stimulated IL-17release into culture medium by ConA is inhibited by OR-13571 andOR-1050. The possibility that inhibition mediated by these two compoundsis due to cytotoxicity was addressed by co-treatment with the RORγagonist OR-12872. OR-12872 alone does not significantly alter IL-17release but it does partly reverse the effects of OR-1050 and OR-13571,suggesting that both compounds act specifically through RORγ.

In a separate study, T-cell blasts were generated from human PBMCaccording to methods previously described (Hoeve, Savage et al. 2006).Frozen T-cell blasts were thawed and cultured for 18 hours in a 24-wellplate with 1×10⁶ cells per well in 700 μL of lymphocyte culture medium.Phorbol 12,13-dibutyrate (500 ng/ml), ionomycin (500 ng/ml) andcompounds (3 μM) were added at the beginning of the culture. Culturemedia were collected and analyzed for hIL-17 level by ELISA. The resultsof treatment with OR-1050 and OR-13571 are shown in FIG. 16C.

Example 7

Methods.

8 to 10-week old male DBA/1 (DBA/1O1aHsd, Harlan Laboratories) mice arehoused in a specific pathogen free (SPF) animal facility. Arthritis isinduced by two injections of collagen subcutaneously in the base of thetail. The initial injection (on day 0) uses bovine type II collagen (2mg/ml from Chondrex, Redmond, Wash.) emulsified in equal volume of CFAcontaining 4 mg/ml of M. tuberculosis (Chondrex). The CII boosterinjection on Day 29 is emulsified in incomplete Freund's adjuvant (IFA).Each animal receives 0.1 ml of emulsion by subcutaneous/intradermalinjection in the tail 2 to 3 cm from the body of the mouse. The boosterinjection site is in the vicinity of but different from the initialinjection site and closer to the body of the animal. OR-1050 wasformulated in HRC-6 as above. On weekdays, the animals received twodoses (a.m. and p.m.) of HRC-6 or 50 mg/kg OR-1050 p.o. (2.5 mls/kg). Onweekends, a single dose of 100 mg/kg was administered (5 mls/kg).

The mice were observed daily for clinical symptoms of CIA based on thefollowing qualitative scale. Each paw was examined individually andscored. Grade 0, normal; grade 1, mild but definite redness and swellingof the ankle or wrist, or apparent redness and swelling limited toindividual digits, regardless of the number of affected digits; grade 2,moderate redness and swelling of ankle or wrist; grade 3, severe rednessand swelling of the entire paw including digits; grade 4, maximallyinflamed limb with involvement of multiple joints. To estimatecumulative disease severity for each animal, an area under the curvescore was calculated for each animal by totaling the sum of the dailyhind paw measurements betweens days 24 and 48. Because of the difficultof adequately distinguishing multiple gradations of change in the frontpaws, scoring of the hind paw only was used in FIG. 17.

Results.

We investigated the effect of the RORγ antagonist OR-1050 on the courseof collagen-induced arthritis in mouse, where T_(H)-17 cells are clearlyinvolved (Courtenay, Dallman et al. 1980; Sato, Suematsu et al. 2006).Animals were treated with OR-1050 (p.o.) starting three days beforeimmunization with collagen type II. The disease phenotype is firstclearly visible about one week after the second injection of CII (at day29), and the disease course is partly suppressed in the OR-1050-treatedanimals when compared to vehicle controls (FIG. 17A). Based on thedifference in overall disease severity determined in the hind limbs ofthe animals (FIG. 17B), the response shows a trend (p<0.09) towards astatistically significant effect.

Example 8

The present invention provides for the discovery of novel small moleculeantagonists to RORγ that are useful in the treatment of autoimmunedisease and other conditions involving the inhibition of T_(H)-17 cellactivity. The steps required to identify a clinical candidate can beoutlined briefly. (1) Novel compounds derived from diverse screeninglibraries, or by directed chemical synthesis from known hits, are testedfor activity in transcriptional and biochemical assays for RORγ. (2)Hits from this primary screening are then characterized for specificityagainst a panel of other nuclear receptors as described above. (3)Candidate lead compounds for more extensive animal studies are selectedfrom these hits on the basis of potency and selectivity. A candidatelead is expected to inhibit murine T_(H)-17 cell formation in culture.The candidate lead molecules are tested for other desirable propertiessuch as bioavailability and metabolic stability that enable therapeuticuse of the compound in animal models of disease. (4) One or more leadRORγ antagonists are tested in animal models of EAE, CIA and IBD whichare known to respond to other agents that inhibit T_(H)-17 function. Ifactive in one or more of these models, the compounds are tested forgeneral immunotoxicity, effects on thymic function and overall immuneresponse, and on general aspects of toxicity such as liver triglyceridecontent and serum lipids known to be affected by ligands to severalnuclear receptors. (5) Such leads are further examined for propertiesthat would prevent use in humans. These include potential for drug-druginteractions and general toxicity in rodents and at least one otherhigher species. If problems in the areas of drug-drug interactions ortoxicity are identified, compounds may be further modified by syntheticmethods, rescreened, and reevaluated to reduce these side effects. Otherfeatures that may be optimized at this stage are bioavailability,chemical stability, and ease of manufacture. (6) One or more candidateclinical compounds are selected for safety studies leading toInvestigative New Drug status granted by the FDA. This step enableshuman clinical trials that will provide evidence for safety andtherapeutic utility. The execution of steps two and beyond is wellunderstood by practitioners of the art of drug discovery. The validationof RORγ antagonist effects in T_(H)-17 cells provides the scientificrationale for undertaking drug development.

As another example, the invention envisages modification of hit or leadmolecules to generate lead candidates. Examples of hits useful forfurther medicinal synthetic work are OR-1050 or OR-133171 describedabove. Analogs of these affect potency at RORγ in a logical manner.Significantly, a representative member of the OR-133171 series was shownto have no measurable transcriptional activity at LXRα. More potentcompounds may be modified by parallel synthetic modification of the corescaffolds of these two compounds in pharmacological assays. These assaysmay identify compounds that increase potency. Other assays for metabolicstability and bioavailability may be introduced in order to identify auseful lead compound. The invention also provides for the discovery ofalternative compound series to those indicated above, in case these failto provide molecules with characteristics suitable for commercialdevelopment, by screening additional small molecule drug-like compoundlibraries.

A drug from the RORγ antagonist class will ideally have good oralbioavailability and a half-life of four hours or more, enabling once ortwice-daily administration. Nuclear receptor ligands have commonly givenrise to orally bioavailable drugs; OR-1050 is an example of anorally-bioavailable RORγ antagonist characterized in these studies. Theadvantages of an orally bioavailable drug are: (i) direct oraladministration is feasible; (ii) unlike injectable biologics, which mayhave an extremely long half life, a small molecule drug with reasonablehalf-life can be withdrawn if necessary to limit side effects and (iii)small molecule drugs are readily manufactured. Such compounds may alsobe selected for clinical development by potency in animal models of EAE,CIA and IBD and by parallel in vivo studies of compound safety. Examplesof useful models for investigation of RORγ antagonist effects on EAE, inaddition to the C57BL/6 mouse immunized with the MOG₃₅₋₅₅ peptide, arefemale SJL/J mice induced with the myelin proteolipid peptide(PLP₁₃₉₋₁₅₁) (Papenfuss, Rogers et al. 2004) and the Lewis rat (Bolton,O'Neill et al. 1997). EAE may also be induced by adoptive transfer ofcultured, antigen-specific T_(H)-17 cells from previously immunized mice(Langrish, Chen et al. 2005). CIA is induced by injection of type 2collagen (Courtenay, Dallman et al. 1980) and this model is recognizedto be IL-23 and T_(H)-17 cell dependent (Murphy, Langrish et al. 2003;Lubberts, Koenders et al. 2004). Mouse models of IBD are now recognizedto have a significant involvement of T_(H)-17 cells (Yen, Cheung et al.2006; Zhang, Zheng et al. 2006; Elson, Cong et al. 2007). Certaingenetic models such as the IL-10^(−/−) mouse (Yen, Cheung et al. 2006)have physiological relevance for understanding disease and have beenshown to require IL-23 for disease induction, but the disease courseitself is prolonged and highly variable in these animals, rendering themunfavorable for pharmacological studies of reasonable duration. On theother hand, transfer of T cell populations with a reduced component of Tregulatory cells to immunodeficient mice will cause disease within about10 weeks (Powrie and Uhlig 2004), a time frame suitable forpharmacological testing. Disease causation in the T cell transfer modelis also dependent on IL-23 (Yen, Cheung et al. 2006). Older mouse IBDmodels that induce damage to the intestinal lining, such as by treatmentwith dextran sodium sulfate (DSS) in drinking water (Spahn, Herbst etal. 2002), are acceptable disease models and may also include asignificant contribution by T_(H)-17 cells.

While the invention has been described with reference to specificexamples, this description is not meant to limit the kind of smallmolecule drug that may be used as part of practicing this invention.

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What is claimed is:
 1. A method of inducing T_(H)-17 celldifferentiation, function or activity in an individual in need thereof,comprising administering to said individual an effective amount of asmall organic molecule agonist of RORγ, wherein said small organicmolecule agonist has a molecular weight of less than about 600 daltons,wherein said small organic molecule agonist has the ability to enhancetranscription from a Gal4 construct comprising a human RORγ ligandbinding domain in a Chinese hamster ovary host cell.
 2. The method ofclaim 1, wherein said T_(H)-17 cell function or activity is release of acytokine.
 3. The method of claim 2, wherein said cytokine isinterleukin-17 or interleukin-22.
 4. The method of claim 1, wherein theRORγ agonist is orally administered.
 5. The method of claim 1 whereinthe RORγ agonist is locally administered.
 6. The method of claim 1,wherein said RORγ agonist is OR-12872 or OR-942.
 7. The method of claim1, wherein the small organic molecule agonist is administered in anamount effective to increase mucosal immunity in the individual.
 8. Themethod of claim 1, wherein the small organic molecule agonist isadministered in an amount effective to increase the immunogenicity of avaccine administered to the individual.
 9. The method of claim 1,wherein the small organic molecule agonist is administered in an amounteffective to increase the number of T cells reactive to an antigen.